EP4646595A1 - Peptide sequencer - Google Patents

Abstract

Peptide sequencing methods are described, in which the non-attached termini of surface end-immobilized peptides are functionalized with a universal docking strand (DS) DNA oligonucleotide. A library of signal molecules (such as fluorophores), each of which is conjugated to an imaging strand (IS) oligo complementary to the DS oligo, is used for characterizing each sequential terminal amino acid of the peptide. Computer assisted analysis is used to identify each amino acid, based on differences in the measured signal that are caused by proximity of the signal molecule(s) to each terminal amino acid.

Description

Filed: January 4, 2024 PEPTIDE SEQUENCER CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and the benefit of the earlier filing of U.S. Provisional Application No. 63/478,661, filed on January 5, 2023, and U.S. Provisional Application No.63/485,904, filed February 18, 2023, both of which are incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] A computer readable text file, entitled "O046-0072PCT_ST26.xml" created on or about January 3, 2024, with a file size of 9,295 bytes, contains the sequence listing for this application and is hereby incorporated by reference in its entirety. FIELD OF THE DISCLOSURE [0003] The present disclosure relates generally to de novo protein and peptide sequencing. Further, it relates to methods of identifying terminal amino acids of a peptide using their interaction with signal molecule(s). BACKGROUND OF THE DISCLOSURE [0004] The flow of genetic information in cells can be described by three fundamental transformations: DNA to DNA (replication), DNA to RNA (transcription), and RNA to protein (translation). Regarding functionality, DNA and RNA determine the structure of proteins, but the ultimate function (or dysfunction in a disease) of a cell is determined by proteins expressed in the cells. Considering how DNA/RNA sequencing platforms have our advanced understanding in general biology, including cancer and other diseases and conditions, knowing exact protein profile of a sample can be beneficial for deeper understanding. Therefore, de novo protein sequencing platforms are needed in disease research for discovering differentiating factors in disease progression and/or novel disease biomarkers. [0005] Currently, untargeted proteomics primarily relies on digesting intact proteins into small peptides and reading their sequence using liquid chromatography-mass spectroscopy (LC-MS). However, LC-MS has some limitations: low sensitivity, high cost, limited dynamic range, and ambiguity in assigning sequences to peptides or peptide fragments, especially to those of the same mass to charge ratio. [0006] De novo peptide sequencing technologies are being developed. Although their final readout methods are different, various companies are employing N-terminal amino acid (NTAA) Filed: January 4, 2024 specific binders for reading out the digested proteins (peptides). As discussed in US 2021/0396762, advances have been made in the field of Digital Analysis of Proteins by End Sequencing (DAPES). For example, in one method surface bound peptides are directly sequenced using a modified Edman degradation step followed by detection, such as with a labeled antibody (WO2010/065531). A modification of DAPES was disclosed, in which single molecule sequencing of peptides is achieved by contacting the peptide with fluorescently labelled N-terminal amino acid binding protein(s) (NAABs), detecting the fluorescence of a NAAB bound to the amino acid, identifying the N-terminal amino acid based on the detected fluorescence, removing the NAAB from the peptide, and repeating with NAABs that bind to different N-terminal amino acids (WO2014/0273004). The N-terminal amino acid is cleaved from the polypeptide by Edman degradation, and the procedure repeated for each newly-exposed N-terminal amino acid. Others teach sequencing of polypeptides using labelled N-terminal amino acids complexing agents, followed by Edman degradation or aminopeptidase cleavage cycles (WO2010/065322); or a method of peptide analysis employing a multi-component detection agent, for instance which includes a first detection agent and second detection agent that, when in proximity, is capable of generating a detectable signal (US 2021/0396762). Other techniques for characterizing proteins include those disclosed in US2003/0138831, US2014/0349860, and WO2013/112745. [0007] Though some of these methods may be promising, each faces significant challenges. Developing specific binders for each amino acid is extremely challenging work. Using covalent dye attachment to amino acids provides read out of only a small subset of amino acids in a peptide sequence. A developing peptide sequence method that uses nanopores for detecting the amino acids, but it suffers from issues with scalability and reliability. Intact protein profiling has also been attempted, but the method relies on an affinity reagent library that does not yet exist. Thus, all the previously available de novo peptide sequencing technologies need significant further development, or even discovery, of either biochemical and/or technological methods to be able to compete with LC-MS. SUMMARY OF THE DISCLOSURE [0008] The advent of next-generation sequencing has greatly accelerated clinical and translational discovery, yet genomic sequencing incompletely portrays the protein landscape of biological systems. Similarly, future de novo protein-sequencing approaches will revolutionize nearly all fields of biological research and medicine. The recent global push towards next- generation protein sequencing has resulted in powerful mass spectrometry and fluorescence- based approaches, however these technologies are unable to completely sequence proteins de Filed: January 4, 2024 novo, suffer from low throughput, and do not have the sensitivity to interpret post-translational modifications with high-fidelity. [0009] Fluorescence lifetime imaging (FLIM) measures single-molecule fluorescence of an individual fluorophore to determine time spent in the excited state before relaxation and emission of a photon. For some fluorophores, the excited state lifetime can be extremely sensitive to local and global environmental changes. Described herein in one embodiment is a single-molecule peptide sequencing methodology using a cyclic Edman-degradation based chemistry with optical readout of fluorescence-lifetime measurements. [0010] Described herein is a new peptide sequencing method, which can be implemented with readily available reagents and equipment. A representative designed workflow is shown in FIGs. 1A-1G. It starts with obtaining unmodified peptides with enzymatic digestion of proteins in the sample, like in LC-MS based workflows. Then, the peptides are attached to a solid substrate from their C-terminus. The N-terminal amino acid of immobilized peptides is functionalized with a universal docking DNA oligo conjugated to a phenyl isothiocyanate (PITC) or a functional equivalent. Here, PITC serves two purposes: (i) it mediates conjugation of the docking oligo; and (ii) it implements cleavage of the N-terminal amino acid (NTAA) when it is time to read out the next amino acid, using Edman degradation. [0011] A library of fluorophores, which are conjugated to an imaging strand (IS) oligo complementary to the docking strand (DS) oligo, is used for determining the N-terminal amino acids. The read out starts with introducing a first fluorophore (conjugated to an IS) for docking (with the DS), and taking a single molecule fluorescence lifetime measurement for each peptide. The fluorescence lifetime of the fluorophore will be different from its free form, since it is in interaction with the NTAA. Furthermore, designed sequence differences in the IS can (optionally) further modulate the fluorescence lifetime readout. Single molecule fluorescence lifetime measurements may be repeated for one or more additional combinations of IS(s) and fluorophore(s) in the library. The N-terminal amino acid (which has been “read”) is then cleaved, for instance using Edman degradation, resulting in the construct ready for the next cycle – analysis of the next amino acid, which is now at the N-terminus of the peptide. Thus, each analysis cycle starts with DS) oligo conjugation and ends with Edman degradation. [0012] Reading out a series of amino acids results in a set of fluorescence lifetime measurement for each amino acid corresponding to each different fluorophore and IS, for as many of each as are used in the analysis. This data can be fed into a machine learning-based prediction algorithm that generates the sequence of all the peptides. [0013] Embodiments of the provided sequence method have myriad benefits. For instance, in provided embodiments there is no need to develop new amino acid-specific binders, which are Filed: January 4, 2024 often difficult to identify (or generate) and validate. All the measurements can be done by using standard fluorophores. Amino acids in various embodiments can be read more than once with different fluorophores and/or DNA imaging strands. This enables high confidence, machine learning-based sequence predictions that employ multiple different data input for each amino acid position. Different imaging oligonucleotide and fluorescent dye designs may be used to further expand variances of fluorescent lifetime readout. [0014] In a provided exemplar embodiment, peptides were bound to a glass surface by the C- terminus, leaving a primary amine at the N-terminus for covalent attachment of a phenyl- isothiocyanate-functionalized oligonucleotide. A complementary imaging strand was hybridized with the docked oligo to bring a fluorophore in close proximity to the N-terminal amino acid and imaged by two-photon FLIM to interrogate the fluorescence lifetime of AF488. Before removal of the N-terminus by Edman degradation, other imaging strands conjugated with BODIPY, KU530- 6, or KU530-R-4 were also used and imaged to interrogate their lifetimes. Of the amino acids tested, tryptophan, arginine, phenylalanine, serine, glutamine, glutamic acid, and phospho-serine showed significant differences in fluorescence lifetime with the illustrated fluorophores. Additionally, amino acids in positions N-1 and N-2 were shown to contribute to lifetime changes. [0015] Provided in one embodiment is a method of sequencing a peptide have an initial NTAA, including sequential interrogation of the initial NTAA using a library of at least two different combinations of a ssDNA DS and a ssDNA IS, where a fluorophore is conjugated to the IS, to produce a set of fluorescence lifetime data having a characteristic fingerprint for each different amino acid of the peptide; wherein each pair of IS and DS are at least partially complementary in sequence. In examples of this embodiment, the sequential interrogation includes detecting and/or measuring interaction between the fluorophore and nucleobase(s) at or near the NTAA by detecting fluorescence lifetime data for each pair IS and DS in the library, for instance using FLIM single-molecule fluorescence measurements. Optionally, these methods of sequencing may also include removing the initial NTAA the peptide by an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. [0016] Also contemplated are method of sequencing a peptide, wherein the method is repeated for each subsequent amino acid in the peptide to produce a matrix of fluorescence lifetime data. Optionally, the data is input into a machine learning algorithm to reconstruct a polypeptide sequence. [0017] Yet another embodiment provides a method for identifying a terminal amino acid (TAA) of a peptide having a N-terminal amino acid (NTAA) and a C-terminal amino acid (CTAA), which method including: binding either the NTAA of the peptide or the CTAA of the peptide to a solid surface to produce a bound TAA; attaching to the non-bound TAA of the peptide a ssDNA docking Filed: January 4, 2024 strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial TAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. [0018] In any of the method embodiments, the library of ISs may include a plurality of ssDNA oligonucleotides that vary so that spatial positioning and/or degrees of freedom of the attached fluorophore, in order to modulate interactions with the NTAA side chain, and thereby modulate the measured fluorescence lifetime. For instance, the library of ISs in some instances includes a plurality of ssDNA oligonucleotides that vary by one or more of: inclusion of a modified nucleotide, inclusion of a non-natural nucleotide, inclusion of a 5’ IS overhang relative to the cognate DS, or inclusion of a 5’ IS underhang relative to the cognate DS. [0019] In various embodiments, the fluorophore is conjugated at an end of the IS. Alternatively, the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide within the IS (FIG.10). [0020] In examples of the provided embodiments, removal of a NTAA is performed under conditions such that the remaining peptide has a new N-terminal amino acid. [0021] Optionally, the peptide(s) to be sequenced is/are immobilized on a solid support. [0022] Yet another embodiment is a method for identifying a NTAA of a peptide, the method including: binding the C-terminal amino acid of the peptide to a solid surface; attaching to the NTAA of the peptide a ssDNA DS; hybridizing to the DS a first ssDNA IS, which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. Optionally, the method may further include cleaving the initial NTAA from the peptide, to leave a next NTAA of the peptide. Optionally, the method further includes repeating the method a plurality of time to identify a sequence of the peptide. [0023] In examples of these method embodiments, cleaving the initial NTAA includes an Edman degradation reaction, enzymatic cleavage or digestion, or a similar process. [0024] Another embodiment is a method of sequencing peptides, which method includes: attaching peptide(s) to be sequenced to a solid substrate by their C-terminus; functionalizing the initial N-terminal amino acid of the immobilized peptides with a universal DS ssDNA oligo; contacting the DSs with an IS oligo complementary to the DS oligo, which IS is conjugated to a Filed: January 4, 2024 first fluorophore; obtaining a single molecule FLIM measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; and cleaving the initial N-terminal amino acid from the peptide to reveal a second N-terminal amino acid; and optionally, carrying out another cycle of analysis for the second N-terminal amino acid. [0025] Also provided are methods of sequence a peptide, essentially as described herein. [0026] Another embodiment is a kit for carrying out any one of the described method embodiments, which kit includes at least one pair of IS and DS. In examples of kit embodiments, the kit includes at least two pairs of IS and DS, where the two pairs differ by the fluorophore contained in the IS or by sequence, or both. [0027] Also provided are databases containing the matrix of fluorescence lifetime data produced by any of the described methods. [0028] Additional embodiments include a method of sequencing a peptide having an initial terminal amino acid (TAA), including: interrogation of the initial TAA using a ssDNA DS attached to the initial TAA and a ssDNA IS to which a signal molecule is conjugated, to produce a measurement of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide; wherein the IS and DS are at least partially complementary in sequence. Optionally, such methods may further include: sequential interrogation of the initial TAA using a library of at least two different combinations of a ssDNA DS and a ssDNA imaging strand (IS), where a signal molecule is conjugated to the IS, to produce a set of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide. In various examples, the initial TAA is: the NTAA of the peptide; or the carboxy-terminal amino acid (CTAA) of the peptide. Optionally, any of these method embodiments can be carried out in parallel on a plurality of peptides. Optionally, the signal molecule may be a fluorophore (such as Alexa Fluor® 488 (AF488), BODIPY-FL, BODIPY-TR, or TAMRA), and optionally the spectral characteristic includes fluorescence lifetime. [0029] Yet another described embodiment is a method of sequencing a peptide having an initial NTAA, the including: sequential interrogation of the initial NTAA using a library of at least two different combinations of a ssDNA DS and a ssDNA IS, where a fluorophore is conjugated to the IS, to produce a set of fluorescence lifetime data having a characteristic measurement for each combination of DS, IS, and fluorophore, wherein each pair of IS and DS are at least partially complementary in sequence. [0030] In any one of the method embodiments, the DS may be a universal DS. [0031] Also provided are peptide analysis (e.g., sequencing) methods in which the sequential interrogation includes detecting and/or measuring interaction between the fluorophore and amino Filed: January 4, 2024 acid sidechain at or near the CTAA or the NTAA by detecting fluorescence lifetime data for each of a plurality of IS / DS pairs in the library. Optionally, in any of the method embodiments, detecting or measuring the interaction includes obtaining FLIM single-molecule fluorescence measurements for each of a plurality of IS / DS pairs in the library. Any of method 1-8 may further including removing the initial CTAA or NTAA of the peptide by an Edman degradation reaction, enzymatic digestion, or a similar process. [0032] The described methods may optionally be repeated for each subsequent amino acid in the peptide, thereby producing a matrix of fluorescence lifetime data. In embodiments, the data is input into a machine learning algorithm to reconstruct a polypeptide sequence. [0033] Also provided herein are peptide analysis methods, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary so that spatial positioning and/or degrees of freedom of the attached fluorophore varies, in order to modulate interactions with the CTAA or NTAA side chain, and thereby modulate the measured fluorescence lifetime. For instance, the library of ISs may include a plurality of ssDNA oligonucleotides that vary by one or more of: inclusion of a modified nucleotide, inclusion of a non-natural nucleotide, inclusion of a 5’ IS overhang relative to the cognate DS, or inclusion of a 5’ IS underhang relative to the cognate DS. Further, the interaction between the CTAA or NTAA in method embodiments may further be influenced by one or more of DS position, degrees of freedom, or another variable described herein. [0034] In any of the method embodiments, the signal molecule (which may optionally be a fluorophore) is conjugated to a nucleotide (such as a modified nucleotide) of the IS. The nucleotide may optionally be at either end (5’ or 3’) of the IS, or somewhere within the IS. [0035] In any of the method embodiments, the peptide is conjugated to a modified nucleotide within (that is, not at the end of) the DS and the fluorophore is conjugated to a modified nucleotide within the IS (as illustrated in FIG.11). [0036] In any of the method embodiments, removal of a CTAA or a NTAA may be performed under conditions such that the remaining peptide has a new terminal amino acid available for another cycle of analysis. [0037] In any of the method embodiments, the peptide may be immobilized on a solid support. [0038] Also provided are databases containing a matrix of signal molecule spectral characteristic data prepared using any of the methods described herein. In certain examples, this data includes measurements of spectral lifetimes of a plurality of different signal molecules, as that lifetime is influenced by proximation of the side chain of different terminal amino acids of analyzed peptides. [0039] Yet another embodiment is a method for identifying a NTAA of a peptide, the method including: binding the C-terminal amino acid of the peptide to a solid surface; attaching to the NTAA of the peptide a ssDNA DS; hybridizing to the DS a first ssDNA IS, which first IS includes Filed: January 4, 2024 a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. Optionally, the method further includes: cleaving the initial TAA from the peptide, to leave a next TAA of the peptide. By way of example, cleaving the initial NTAA may include an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. Optionally, the method is repeated a plurality of time to identify a sequence of the peptide. [0040] A further embodiment is a method for identifying a CTAA of a peptide, the method including: binding the N-terminal amino acid of the peptide to a solid surface; attaching to the CTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA IS, which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. Optionally, the method further includes cleaving the initial TAA from the peptide, to leave a next TAA of the peptide. By way of example, cleaving the initial NTAA may include an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. Optionally, the method is repeated a plurality of time to identify a sequence of the peptide. [0041] Also provided are methods of sequencing peptides, which methods include: attaching peptide(s) to be sequenced to a solid substrate by their C-terminus; functionalizing the initial N- terminal amino acid of the immobilized peptides with a universal DS ssDNA oligo; contacting the DSs with an IS oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule FLIM measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; and cleaving the initial N-terminal amino acid from the peptide to reveal a second N-terminal amino acid; and optionally, carrying out another cycle of analysis for the second N-terminal amino acid. [0042] Also provided are methods of sequencing peptides, which methods include: attaching peptide(s) to be sequenced to a solid substrate by their N-terminus; functionalizing the initial C- terminal amino acid of the immobilized peptides with a universal DS ssDNA oligo; contacting the DSs with an IS oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule FLIM measurement for the first fluorophore for each peptide; Filed: January 4, 2024 optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; and cleaving the initial C-terminal amino acid from the peptide to reveal a second C-terminal amino acid; and optionally, carrying out another cycle of analysis for the second C-terminal amino acid. [0043] Another embodiment is a method of sequencing a peptide, essentially as described herein. Contemplated in this embodiment are methods that include detecting at least one spectral characteristic of a signal molecule, where the spectral characteristic is not fluorescence lifetime. [0044] Yet another provided embodiment is a kit for carrying out the method of any of provided embodiments, which kit includes at least one pair of IS and DS. For instance, examples of such kits include at least two pairs of IS and DS, where the two pairs differ by the signal molecule (e.g., fluorophore) attached to the IS, or by sequence, or both. [0045] Also described herein are compounds having the Formula (I) or a salt, or solvate thereof, independently is selected from the group consisting of C1-C6 alkyl, -NO2, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO3, or any other common electron withdrawing group; R¹ and R² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, -O-(C₁-C₆ alkyl), C1-C6 alkyl, hydroxy, halogen, -O- alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, halogens, -O-alkyl, -S-alkyl, -O-C(=O)R, - N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating group. In examples, x is 0; and/or y is 0; and/or the C1-C6 alkyl of R¹ or R² is methyl, and the -O- (C1-C6 alkyl) of R¹ or R² is methoxy. Filed: January 4, 2024 [0046] Additional provided compounds have the Formula (II) or a salt, or solvate x is 0, 1 or 2; each R group of C1-C6 alkyl, -NO2, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO3, or any other common electron withdrawing groups; R¹ and R² are independently selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, -O-(C1-C6 alkyl), C1-C6 alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)- R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, - N-(C=O)-OR, or any other common electron donating groups. In examples, x is 0; and/or y is 0; and/or the C₁-C₆ alkyl of R¹ or R² is methyl, and the -O-(C₁-C₆ alkyl) of R¹ or R² is methoxy.

Filed: January 4, 2024 [0047] Additional provided compound embodiments have the structure: ; wherein R₁ and R₂ are and OCH₃; with the proviso that R1 and R2 are the same; or a salt or solvate thereof. For instance, one example such compound of embodiment 40, which is (4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. [0048] Also provided is a process for preparing a compound of Formula (I) or a salt or solvate thereof, of Formula (III) Filed: January 4, 2024 to a compound a Formula (II) or , and thereafter or solvate thereof to the compound of Formula (I) or a salt or solvate thereof, wherein: x is 0, 1 or 2; each R independently is selected from the group consisting of C1-C6 alkyl, -NO2, halogen, -C=OR, -C=SR, -C=ONR, - C=OOR, -SO3, or any other common electron withdrawing group; R¹ and R² are independently selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, -O-(C1-C6 alkyl), halogen, - O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating group; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, halogens, -O-alkyl, -S-alkyl, -O-C(=O)R, - N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating Filed: January 4, 2024 group. In examples of this embodiment, the compound of Formula (III) or the salt or solvate thereof is first converted to a compound of Formula (IV) or solvate thereof, followed by conversion of the or the salt or solvate thereof to the compound of Formula (II) or solvate thereof. In examples, of these process or embodiments, the conversion of the compound of Formula (III) of the salt or solvate thereof to the compound of Formula (IV) or the salt or solvate thereof takes place by reacting carbon disulfide (CS2) with the compound of Formula (III). By way of example, the reaction takes place in the presence of a base (such as a (C1-C6 alkyl)3N, or more particularly triethylamine). [0049] Also provided are examples of this process embodiment, wherein the conversion of compound of Formula (IV) or the salt or solvate thereof to the compound of Formula (II) or the salt or solvate thereof takes place by reacting the compound of Formula (IV) or the salt or solvate thereof with di-tert-butyl carbonate (O-(C(=O)-OC(CH3)2)2). By way of example, the reaction may take place in the presence of one or more bases, such as the one or more bases include dimethyl aminopyridine (DMAP) and triethylamine.

Filed: January 4, 2024 [0050] Also described are process embodiments, wherein the compound has the structure: ; wherein R₁ and R₂ are and OCH₃; with the proviso that R1 and R2 are the same; or a salt or solvate thereof. For instance, the compound in some examples is (4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. [0051] Also provided are use of any of the described compounds in any of the method embodiments of peptide analysis as described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0052] FIGs.1A-1G is a schematic of an embodiment of a fluorescence lifetime imaging (FLIM)- based peptide sequencing workflow. The illustrated approach allows for sequential interrogation of the N-terminal amino acid (NTAA) by a library of different combinations ssDNA imaging strands (ISs) and fluorophores to produce a set of fluorescence lifetime data with a characteristic fingerprint for each different amino acid. The NTAA is removed by Edman degradation and the method is repeated for one or more subsequent amino acids to yield a matrix of fluorescence lifetime data. This data can be input into a machine learning algorithm to reconstruct the polypeptide sequence. [0053] FIG.2 shows single stranded DNA (ssDNA) docking and imaging strand sequences and schematic arrangement. In embodiments, the ssDNA imaging strands (IS) can be modified in a variety of ways, such as inclusion of modified or non-natural nucleotides, inclusion of a 5’ IS overhang relative to the docking strand (DS), or inclusion of a 5’ IS underhang relative to the DS. These variables adjust the spatial position and/or degrees of freedom of the attached fluorophore, Filed: January 4, 2024 and thereby modulate interactions with the NTAA side chain, which modulates the measured fluorescence lifetime. This enables identification of each NTAA. Imaged in the lower panel of FIG. 2 are DS1 (SEQ ID NO: 1), IS1 (SEQ ID NO: 2), IS2 (SEQ ID NO: 3), and IS3 (SEQ ID NO: 4). [0054] FIG.3 illustrates chemical structure examples of a bifunctional linker precursor (top left) and product, known as maleimidophenyl isothiocyanate (MPITC) (IUPAC: 1-(4- (isothiocyanatophenyl)-1H-pyrrole-2,5-dione) (bottom left); a DNA DS–peptide conjugate (middle); and a DNA IS–fluorescent dye conjugate (right). In the illustrated example, the DNA DS has a 3’ propylthiol modification that enables conjugation to the maleimido group of the MPITC linker. The peptide is conjugated to the isothiocyanato group of the MPITC linker. In the illustrated example, the DNA IS has a 5’ hexylamine modification for conjugation to a fluorophore modified with a chemical crosslinker, such as a maleimido group. [0055] FIG.4 is a bar graph illustrating fluorescent lifetimes of AF488 and BODIPY-FL conjugated to imaging strand 1 (“IS1”) in proximity to various synthetic peptides containing differing N-terminal amino acids as shown on the X-axis (format: N-terminus-AA1-AA2-AA3). This figure illustrates that using different fluorophores enables differentiation of sequent amino acids, for instance as shown for FGG, SGG, and RGG. [*, p<0.05; ***, p<0.005; Two-way ANOVA with a Tukey post hoc; N= multiple fields of view within 3-8 samples]. [0056] FIG.5 is a bar graph that illustrates the mean normalized fluorescence intensity measured from various parts of the embodiment (e.g., various assemblies of the complete workflow). Three sub structures (silane-PEG; silane-PEG+Peptide+MPITC+DS; silane-PEG+DS+IS) and the complete embodiment were analyzed. A peptide containing a serine at the N-terminus and imaging strand 1 (“IS1”) conjugated to AF488 was used in the analysis. These data in the figure indicate that the collected fluorescence lifetime data is dominated by the fluorophores and suggests that other components of the construct do not produce significant fluorescence. Data was normalized to blank samples [*, p>0.05; ***, p<0.005; ****, p<0.0001; One-way ANOVA with Tukey Post hoc; N = 3 samples]. [0057] FIG.6 is a bar graph that illustrates interrogation of post-translationally modified (PTM) N- terminal amino acids. Fluorescence lifetime of four separate fluorophores (AlexaFluor® 488, BODIPY-FL, BODIPY-TR, and TAMRA) conjugated to imaging strand IS1, in proximity to N- terminal amino acids containing either serine (S) or a common post-translational modified phosphor-serine (PhosS). [N=1 sample]. [0058] FIGs.7A-7C illustrate normalized fluorescence lifetimes from fluorophores conjugated to IS1, before and after removal of the NTAA (position “N”) to expose the next amino acid in the polypeptide chain (position “N-1”). In the illustrated embodiment, Edman degradation is used to remove the NTAA. FIG. 7A. Fluorescence lifetimes measured from imaging strand 1 (IS1) Filed: January 4, 2024 conjugated with Alexa Fluor 488 (IS1-AF488) with peptides before and after single Edman degradation to remove the N-terminal amino acid at position “N” and expose the next amino acid in the polypeptide chain (“N-1”). The cleaved amino acid in the sequence is shown in parentheses. [N= multiple fields of view within 3 samples]. FIG. 7B. Fluorescence lifetimes measured from imaging strand 1 (IS1) conjugated with BODIPY-FL (IS1-BODIPY-FL) with peptides before and after single Edman degradation to remove the N-terminal amino acid at position “N” and expose the next amino acid in the polypeptide chain (“N-1”). The cleaved amino acid in the sequence is shown in parentheses. [*, p<0.05; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples].The parentheses in the N-terminal sequence (X-axis label) indicate the amino acid that was removed by Edman degradation. Data are normalized to a free (unannealed) IS1- fluorophore control. FIG.7C illustrates a full cycle of terminal-amino acid analysis, including reset to the second terminal amino acid; different fluorophores are indicated by shape of the illustrated star. [0059] FIGs.8A-8H are a series of bar graphs showing normalized fluorescence lifetimes from fluorophores conjugated to IS1, before and after multiple Edman degradation cycles to sequentially remove and collect the lifetime information from amino acids in the polypeptide chain. IS1-AF488 is shown in FIGs.8A-8D and IS1-BODIPY-FL is shown in FIGs.8E-8H. Fluorescence lifetimes were collected from fluorophores adjacent to peptides containing tryptophan (FIGs.8A & 8B and FIGs.8E & 8F) as well as arginine (FIGs.8C & 8D and FIGs.8G & 8H) in the second (“N-1”) and third (“N-2”) positions. FIG. 8A. Fluorescence lifetimes measured from imaging strands conjugated with Alexa Fluor 488 with peptides that possessed a tryptophan at the second position along the peptide (“N-1”) before and after multiple Edman degradation cycles. [***, p<0.005; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG. 8B. Fluorescence lifetimes measured from imaging strands conjugated with Alexa Fluor 488 with peptides that possessed a tryptophan at the third position along the peptide (“N-2”) before and after multiple Edman degradation cycles. [*, p<0.05; **, p<0.01; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG.8C. Fluorescence lifetimes measured from imaging strands conjugated with Alexa Fluor 488 with peptides that possessed an arginine at the second position along the peptide (“N-1”) before and after multiple Edman degradation cycles. [N= multiple fields of view within 3 samples]. FIG. 8D. Fluorescence lifetimes measured from imaging strands conjugated with Alexa Fluor 488 with peptides that possessed an arginine at the third position along the peptide (“N-2”) before and after multiple Edman degradation cycles. [*, p<0.05; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG.8E. Fluorescence lifetimes measured from imaging strands conjugated with BODIPY-FL with peptides that possessed a tryptophan at the second position along the peptide (“N-1”) before and after Filed: January 4, 2024 multiple Edman degradation cycles. [*, p<0.05; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG.8F. Fluorescence lifetimes measured from imaging strands conjugated with BODIPY-FL with peptides that possessed a tryptophan at the third position along the peptide (“N-2”) before and after multiple Edman degradation cycles. [*, p<0.05; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG. 8G. Fluorescence lifetimes measured from imaging strands conjugated with BODIPY-FL with peptides that possessed an arginine at the second position along the peptide (“N-1”) before and after multiple Edman degradation cycles. [*, p<0.05; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. FIG. 8H. Fluorescence lifetimes measured from imaging strands conjugated with BODIPY-FL with peptides that possessed an arginine at the third position along the peptide (“N-2”) before and after multiple Edman degradation cycles. [*, p<0.05; **, p<0.01 One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. [0060] FIG.9 is a series of bar graphs that illustrate dependence of fluorescence lifetime on the combined choice of DNA IS sequence and fluorescent dye. The illustrated data was obtained using the same peptides, but with different Imaging Strands (IS1 (SEQ ID NO: 2), IS2 (SEQ ID NO: 3), and IS3 (SEQ ID NO: 4)) and fluorophores (Alexa Fluor® 488 in the top panel; BODIPY in the bottom). IS1 and IS2 have higher melting temperatures, and IS3 has single base overhang. Data are normalized to the fluorescence lifetime of the respective free fluorescent dye. [N=1 sample] [0061] FIGs. 10A-10C illustrate generation of unique peptide fingerprints using provided methods. FIG. 10A is a two-dimensional graph showing a fluorescent lifetime fingerprint of different N-terminal amino acids. FIG. 10B illustrates that fluorophore cycling creates a robust amino acid fingerprint. FIG.10C illustrates that complex multivariate results increase accuracy of neural network prediction. Fluorescent lifetime fingerprint of different N-terminal amino acids is illustrated, with a theoretical neural network approach for the identification of the amino acids. [0062] FIG.11 illustrates an alternative peptide sequencing system embodiment, using a DNA major groove design. The immobilized peptide is conjugated to a modified nucleotide within the DS (that is, not immediately proximal to either end of the DS) and the fluorophore is conjugated to a modified nucleotide within the IS (that is, not immediately proximal to either end of the IS). This provides additional tunable control over the interaction between the fluorescent dye and structural components of the DNA docking strand:imaging strand (DS:IS) complex. In the current embodiment, there is interaction between the fluorescent dye and the nucleobases of the blunt end of the DS:IS complex, whereas, in this alternative embodiment, the fluorescent dye is conjugated internally and therefore cannot access the blunt end of the complex. This has been verified with molecular dynamics simulations in silico. A peptide is conjugated to a modified Filed: January 4, 2024 nucleotide within the docking strand and the fluorophore is conjugated to a modified nucleotide within the imaging strand. [0063] FIG. 12 is a bar graph depicting the native fluorescence lifetimes measured from fluorophore-conjugated imaging strands (IS1) in water. [*, p<0.05; **, p<0.01; ****, p<0.001; One- way ANOVA with a Tukey post hoc; N= multiple fields of view within 3 samples]. [0064] FIG.13 is a bar graph which illustrates the various fluorescence lifetimes measured from various imaging strands (“IS1”) containing long-lifetime fluorophores, KU530-6 and KU530-R-4 to identify the N-terminal amino acids. [**, p<0.01; ****, p<0.001; Two-way ANOVA with a Tukey post hoc; N= multiple fields of view within 3-4 samples]. [0065] FIG.14 Fluorescence lifetimes measured from imaging strands conjugated with AF488, BODIPY-FL, or KU530-6 with peptides that possessed common post-translational modifications of either phosphorylation on serine or acetylation of lysine at the N-terminus. [*, p<0.05; One-way ANOVA with Tukey Post hoc; N=multiple fields of view within 3 samples]. [0066] FIG. 15 Fluorescence lifetimes reported from IS1 conjugated with AF488 in control samples of SGG as well as post-translationally modified serine (PhosSGG). Using a phosphatase, the phosphorylation was removed from the PhosSGG and FLIM of IS1-AF488 was conducted and reported similar lifetimes to the control. [*, p<0.05; One-way ANOVA with Tukey Post hoc; N = multiple regions within 3 samples]. [0067] FIG.16 Normalized lifetime “heatmap” from sequenced data as reported from 4 separate fluorophore-conjugated imaging strands with the peptide screening experiments. Each data was normalized to GGGS for each fluorophore and patterned based on the corresponding range of normalized lifetimes. [0068] FIG. 17 Fluorescence lifetimes measured from imaging strands conjugated with Alexa Fluor 488 with peptides that possessed a tryptophan or arginine at the second and third positions along the peptide (“N-2”) to determine if amino acids at N-1 or N-2 affect the lifetime of the fluorophore. [N= multiple fields of view within 3 samples]. [0069] FIG.18 Fluorescence lifetimes measured from imaging strands conjugated with BODIPY- FL with peptides that possessed a tryptophan or arginine at the second and third positions along the peptide (“N-2”) to determine if amino acids at N-1 or N-2 affect the lifetime of the fluorophore. [N= multiple fields of view within 3 samples]. [0070] FIG.19 Fluorescence lifetimes measured from imaging strands conjugated with KU530-6 with peptides that possessed a tryptophan or arginine at the second and third positions along the peptide (“N-2”) to determine if amino acids at N-1 or N-2 affect the lifetime of the fluorophore. [*, p<0.05; ***, p<0.005; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. Filed: January 4, 2024 [0071] FIG. 20 Fluorescence lifetimes measured from imaging strand 1 (IS1) conjugated with KU530-6 with peptides before and after single Edman degradation to remove the N-terminal amino acid at position “N” and expose the next amino acid in the polypeptide chain (“N-1”). The cleaved amino acid in the sequence is shown in parentheses. [**, p<0.01; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. [0072] FIGs.21A-21C Sequencing data from a more elaborate synthetic peptide which contains the full sequence of WGRSGGSDC (FIG.21A; SEQ ID NO: 5), RGWSGGSDC (FIG.21B; SEQ ID NO: 6), and WRGSGGSDC (FIG. 21C; SEQ ID NO: 7). The full workflow was completed, including imaging strand cycles and multiple Edman degradation cycles. As shown, fluorescence lifetimes were measured from imaging strands conjugated with Alexa Fluor 488 at each step of the workflow. The parentheses designate amino acids which have been cleaved during the Edman degradation process. [FIG.21A: **, p<0.01; ***, p<0.005; ****, p<0.001 One-way ANOVA, Tukey post hoc; FIG.21B: *, p<0.05; **, p<0.01; ****, p<0.001 One-way ANOVA, Tukey post hoc; FIG.21C: *, p<0.05; ****, p<0.001, One-way ANOVA, Tukey post hoc; N=multiple fields of view within 3 samples for all panels]. [0073] FIGs.22A-22C Sequencing data from a more elaborate synthetic peptide which contains the full sequence of WGRSGGSDC (FIG.22A; SEQ ID NO: 5), RGWSGGSDC (FIG.22B; SEQ ID NO: 6), and WRGSGGSDC (FIG. 22C; SEQ ID NO: 7). The full workflow was completed, including imaging strand cycles and multiple Edman degradation cycles. As shown, fluorescence lifetimes were measured from imaging strands conjugated with BODIPY-FL at each step of the workflow. The parentheses designate amino acids which have been cleaved during the Edman degradation process. [FIG. 22B: *, p<0.05; ****, p<0.001 One-way ANOVA, Tukey post hoc; N=multiple fields of view within 3 samples for all panels]. [0074] FIGs.23A-23C Sequencing data from a more elaborate synthetic peptide which contains the full sequence of WGRSGGSDC (FIG.23A; SEQ ID NO: 5), RGWSGGSDC (FIG.23B; SEQ ID NO: 6), and WRGSGGSDC (FIG. 23C; SEQ ID NO: 7). The full workflow was completed, including imaging strand cycles and multiple Edman degradation cycles. As shown, fluorescence lifetimes were measured from imaging strands conjugated with KU530-6 at each step of the workflow. The parentheses designate amino acids which have been cleaved during the Edman degradation process. [FIG.23A: *, p<0.05; **, p<0.01; ****, p<0.001 One-way ANOVA, Tukey post hoc; FIG.23B: *, p<0.05; **, p<0.01 One-way ANOVA, Tukey post hoc; N=multiple fields of view within 3 samples for all panels]. [0075] FIGs.24A-24C Sequencing data from a more elaborate synthetic peptide which contains the full sequence of WGRSGGSDC (FIG.24A; SEQ ID NO: 5), RGWSGGSDC (FIG.24B; SEQ ID NO: 6), and WRGSGGSDC (FIG. 24C; SEQ ID NO: 7). The full workflow was completed, Filed: January 4, 2024 including imaging strand cycles and multiple Edman degradation cycles. As shown, fluorescence lifetimes were measured from strands conjugated with KU530-R-4 at each step of the workflow. The parentheses designate amino acids which have been cleaved during the Edman degradation process. [N=multiple fields of view within 3 samples for all panels]. [0076] FIG. 25 Normalized lifetime “heatmap” from sequenced data as reported from four separate fluorophore-conjugated imaging strands. Each data was normalized to GGGS for the respective fluorophore and patterned based on the corresponding range of normalized lifetimes. Peptides shown are SEQ ID NO: 5 (top), SEQ ID NO: 6 (middle) and SEQ ID NO: 7 (bottom). [0077] FIG.26 Fluorescence lifetimes measured from various KU dyes with different peptides. [*, p<0.05; ***, p<0.005; ****, p<0.001; One-way ANOVA with Tukey post hoc; N=multiple fields of view within 3 samples] REFERENCE TO SEQUENCES [0078] The nucleic acid and/or amino acid sequences described herein are shown using standard letter abbreviations, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate. [0079] SEQ ID NO: 1 shows the nucleic acid sequence of an exemplary Docking Strand DS1: ATCTACATATCTC. [0080] SEQ ID NO: 2 shows the nucleic acid sequence of a first exemplary Imaging Strand IS1: TAGATGTATAGAG. [0081] SEQ ID NO: 3 shows the nucleic acid sequence of a second exemplary Imaging Strand IS2: TLALGATGTATAGAG (where “L” indicates the nucleic acid is locked). [0082] SEQ ID NO: 4 shows the nucleic acid sequence of a third exemplary Imaging Strand IS3: TTLALGATGTATAGAG (where “L” indicates the nucleic acid is locked). [0083] SEQ ID NO: 5 shows the amino acid sequence of a synthetic peptide: WGRSGGSDC [0084] SEQ ID NO: 6 shows the amino acid sequence of a synthetic peptide: RGWSGGSDC [0085] SEQ ID NO: 7 shows the amino acid sequence of a synthetic peptide: WRGSGGSDC [0086] SEQ ID NO: 8 shows the amino acid sequence shared between SEQ ID NOs: 5-7: (XXX)SGGSDC DETAILED DESCRIPTION [0087] The advent of next-generation sequencing has greatly accelerated clinical and translational discovery, yet genomic sequencing incompletely portrays the protein landscape of biological systems. Similarly, future de novo protein-sequencing approaches will revolutionize Filed: January 4, 2024 myriad fields of biological research and medicine. The recent global push towards next-generation protein sequencing has resulted in powerful mass spectrometry and fluorescence-based approaches, however these technologies are unable to completely sequence proteins de novo, suffer from low throughput, and do not have the sensitivity to interpret post-translational modifications with high-fidelity. [0088] In this disclosure, methods are described for determining the terminal amino acid(s) of peptides. In embodiments, this is accomplished by distinguishing changes in the fluorescence lifetime of common fluorophores upon their interaction with each terminal amino acid. Using described techniques to attach fluorophores to DNA oligos, the fluorophore is strategically placed in close proximity to an amino acid and the fluorescence lifetime based on the fluorophore-amino acid interaction is collected. Additionally, the use of oligos expands the characterization of amino acids within the sequence, as various fluorophores can be attached to collect different fluorescence lifetime information. In addition, changes in the imaging strand oligo properties can be included to alter the fluorophore-amino acid interaction, which can be helpful for instance in characterizing post-translational modified (PTM) amino acids. Multiple terminal amino acid sequence removal cycles (for instance, Edman degradation cycles) can be performed with described current workflows to enable sequencing of the peptide. [0089] Described herein is a single-molecule peptide sequencing methodology, embodiments of which use a cyclic Edman-degradation based chemistry with an optical readout, for instance of fluorescence-lifetime measurements. Fluorescence lifetime imaging (FLIM) measures single- molecule fluorescence of an individual fluorophore to determine time spent in the excited state before relaxation and emission of a photon. For some fluorophores, the excited state lifetime can be extremely sensitive to local and global (but molecular-scale) environmental changes. [0090] In an exemplary described workflow, peptides were bound to a glass surface by the C- terminus, leaving a primary amine at the N-terminus for covalent attachment of a phenyl- isothiocyanate-functionalized oligonucleotide (the docking strand, DS). A complementary imaging strand (IS) was hybridized with the docked DS oligo to bring a fluorophore (attached to the IS) in close proximity to the N-terminal amino acid, and the system was imaged by two-photon FLIM to interrogate the fluorescence lifetime of the fluorophore (e.g., AF488). Before removal of the N- terminus by Edman degradation, the lifetime of a second fluorophore-labeled IS (specifically, a BODIPY-FL imaging strand) was also measured. Of the amino acids tested, tryptophan, arginine, phenylalanine, serine, and phospho-serine showed significant differences in fluorescence lifetime. Additionally, it was demonstrated that amino acids in positions N-1 and N-2 contributed to lifetime changes. This technology enables a high sensitivity, high throughput method to sequence peptides. Filed: January 4, 2024 [0091] Aspects of the current disclosure are now described with additional details and options as follows: (I) Overview of Polypeptide Sequencing Methods; (II) Selection & Preparation of Peptides for Analysis; (III) Blocking Undesirable Chemical Reactivity with Protecting Groups; (IV) Support Surface for Immobilization of Peptides; (V) Support Surface Conjugation of Peptides; (VI) Modification of Free End of Immobilized Peptides; (VII) Signal Molecules; (VIII) Imaging Strands, and Attachment of Signal Molecules; (IX) Docking Strands, and Attachment of Docking Strand to Immobilized Peptides; (X) Binding of Labeled Imaging Strand to Docking Strand on Immobilized Peptides; (XI) Detection of Imaging Strand Signals; (XII) Cleavage of Terminal Amino Acid and Release of Docking Strand; (XIII) Cyclic Method and Applications; (XIV) Assembly of Sequence – Comparison to Database(s); (XV) Kits and Articles of Manufacture; (XVI) Representative Definitions; (XVII) Exemplary Embodiments; (XVIII) Experimental Examples; (XV) Select References; and (XVI) Closing Paragraphs. These headings do not limit the interpretation of the disclosure and are provided for organizational purposes only. (I) Overview of Polypeptide Sequencing Methods [0092] This disclosure presents new peptide sequencing methods, which can be implemented with readily available and herein described reagents and equipment. The designed workflow of one embodiment can be seen in FIGs.1A-1G. It starts with obtaining unmodified peptides that have been enzymatically digested from whole proteins (much as would be performed in LC-MS based peptide sequencing workflows). The enzymatic digestion cleaves proteins. These digested (or otherwise fragmented) peptides are attached to a solid substrate from their c-terminus (FIG. 1A) via, for instance, thiol-maleimide click chemistry between the thiol of the cysteine residue at the c-terminus and the maleimide of a silane-PEG-maleimide linker that had been previously conjugated to the glass surface. [0093] The n-terminus of immobilized peptides is functionalized with a docking strand DNA oligo (which is optionally a universal docketing strand), conjugated to phenyl isothiocyanate (PITC) as can be seen in FIG.1B. Here, PITC serves two different purposes: 1) it mediates the conjugation of the docking oligo and 2) it permits cleavage of the N-terminal amino acid when it is time to read out the next amino acid by using Edman degradation (Smith, In: Encyclopedia of Life Sciences. 2001, MacMillan Publishers Ltd, Nature Publishing Group, available online at els.net.). A fluorophore (or another signaling molecule) is conjugated to a complementary oligo, termed the imaging strand (IS), and is used to place the fluorophore in close proximity to the N-terminal amino acid for direct interaction (FIG.1C). In this embodiment, a library of fluorophores can be attached the imaging strands to measure various fluorophore lifetimes (or other signal molecules, and other Filed: January 4, 2024 spectral characteristic(s)) as well as different interactions with then-terminal amino acids. Additionally, imaging strands can be varied to alter the distance between fluorophore and n- terminal amino acid, ultimately changing the fluorescence lifetime readouts and providing more data to separately evaluate each amino acid. [0094] The read out starts with bringing in the first fluorophore (attached to an IS) for docking with the DS and collecting a single molecule fluorescence lifetime measurement for each peptide (FIG. 1D). As depicted in FIG.1E, the fluorescence lifetime of the fluorophore will be different from its free form since it is in interaction with the n-terminal amino acid and vary between different fluorophores (Anju et al., ACS Omega 4(7):12357-12565, 2019; US Patent No.7,046,661). Single molecule FLIM may be repeated for the other fluorophores in the IS library (FIGs.1C-1E). Finally, the N-terminal amino acid is cleaved, for instance via Edman degradation, and the immobilized peptide is then ready for the next cycle (FIG.1F). The next cycle starts from DS oligo conjugation to the new n-terminal amino acid (FIG. 1B), performing FLIM measurements with various ISs (FIGs. 1C-1E) and ends with another (Edman) degradation (FIG. 1F) releasing the N-1 TAA (compared to the initial TAA). [0095] Reading out the amino acids of the peptide (for instance, all the amino acids of the peptide) results in a set of fluorescence lifetime measurement for each amino acid corresponding to different fluorophores. This data can be processed in a machine learning based prediction algorithm that will generate the sequence of the peptides (FIG. 1G). To improve prediction performance, a training data set that has measurements of known peptide sequences can be provided, and referenced. It is expected this method will enable read out of millions of peptides each, containing 10-20 or more amino acids, analyzed in parallel. [0096] In representative embodiments, C-terminal carboxylic acid (or a side chain of amino acid, such as the sulfhydryl of a cysteine residue) of a natural, synthetic, or modified peptide (or a collection of two or more peptides) is first conjugated to a supporting surface (FIGs.1A-1G). In examples, such peptide(s) can be prepared through fragmentation of proteins, such as enzymatic degradation of proteins through treatment with one or more proteases (e.g., peptidases and/or proteinases). Alternatively, the protein can be chemically digested with agents such as cyanogen bromide. [0097] Before or after enzymatic or chemical digestion, reactive chemical groups of the protein or peptide, such as the side chains functional groups of lysine (amino), aspartic acid (carboxyl), glutamic acid (carboxyl), cysteine (sulfhydryl), serine (hydroxyl), threonine (hydroxyl), tyrosine (hydroxyl), and arginine (guanidino), can be blocked using a variety of known chemical reactions. This prevents the reaction-labile side chains of these amino acids from interfering with subsequent chemical reactions of the workflow. Filed: January 4, 2024 [0098] Surface conjugation of the peptide(s) may be achieved by using a bifunctional DBCO- tetraethylene glycol-maleimide crosslinker to attach to an azide-functionalized glass surface (or more generally, a functionalized support surface). The maleimido group of the crosslinker reacts with the sulfhydryl group of a C-terminal cysteine residue of a synthetic peptide to form a covalent bond and the DBCO group of the crosslinker reacts with an azido group on the support surface to form a covalent bond. In a second example, a maleimide-functionalized glass surface is prepared, and the sulfhydryl group of a cysteine residue contained within a synthetic peptide is covalently bonded to the maleimide. [0099] Subsequently, in one example, the N-terminal amine of the peptide is conjugated covalently to the isothiocyanato group of a bifunctional maleimidophenyl isothiocyanate (MPITC) crosslinker (FIG. 3). Then, a designed synthetic single stranded DNA (ssDNA) docking strand (DS) of a chosen sequence (FIG.2) with a modification on the 3’ end of the oligonucleotide, which in one example is an alkyl thiol group such as (3-mercaptopropyl)phosphate, is covalently linked to the N-terminal maleimido group of the surface-conjugated MPITC-peptide construct (FIGs.1 and 3). The DS can be universal, in that it has can be bound to any/all peptides in an experiment regardless of the primary sequence of the peptides. [0100] To this synthetic ssDNA DS, a ssDNA imaging strand (IS) with a 5’ conjugated fluorophore is annealed to form a double stranded DS:IS complex. The designed ssDNA IS of a chosen sequence with a modification on the 5’ end of the oligonucleotide. In one example, this modification is an alkyl amine such as (6-aminohexyl)phosphate, which is conjugated covalently to the maleimido group of a maleimide-functionalized fluorophore (FIGs. 2 and 3). The full assembly of the peptide-DS conjugate with an annealed IS-fluorophore (including fluorophores such as Alexa Fluor® 488 [AF488] and BODIPY-FL) have been modelled and simulated in silico to guide the design of oligonucleotide sequences and the design of the various linkers within the complete assembly. The length of the IS may be influenced by the melting temperature of the DS:IS complex. Designing IS with certain guanine and cytosine content and adjusting environmental salt conditions can assist in producing the desired IS. The minimal length of IS may be 8-10 base pairs. The maximum length of IS may be influenced (e.g., constrained) by the upper range of the melting temperature of the DS:IS complex. [0101] Upon forming the DS:IS-fluorophore complex, the fluorophore linked to the 5’ end of the IS oligonucleotide is positioned in close spatial proximity to the side chain of the NTAA of the peptide (FIGs. 1 and 2). This close spatial proximity facilitates weak molecular interactions between the fluorophore and the NTAA. Based on the chemical structure of the NTAA side chain and the chemical structure of the chosen fluorophore, a change in the measured fluorescence lifetime of the fluorophore is observed relative to the fluorescence lifetime of free fluorophore. Filed: January 4, 2024 Furthermore, the ssDNA IS sequence can contain a variety of synthetic structural features, including modified or non-natural nucleotides, a 5’ single stranded overhang of one or more nucleotides, or a 5’ underhang of one or more nucleotides (FIG.2). In one example, locked nucleic acids (LNA), also known as locked nucleotides, can be included at the 5’ end of the IS oligonucleotide sequence. LNAs exhibit stronger base pairing to their complementary nucleic acids. In this case, inclusion of LNA increases the stability of the DS:IS-fluorophore complex and constrains the degrees of freedom of the fluorophore by preventing the terminal base pairing from being temporarily disrupted in a stochastic manner. These synthetic structural features of the IS affect the positioning and degrees of freedom of the fluorophore, which alters the relative positioning of the fluorophore and NTAA sidechain and results in a differential change in measured fluorescence lifetime of the fluorophore. Therefore, a unique fluorescence lifetime signature is acquired for the NTAA based on the sequence design of the IS and the choice of the conjugated fluorophore. [0102] The noncovalent bonding of the DS:IS-fluorophore complex is then disrupted, so that the IS-fluorophore can be removed, and a new IS-fluorophore molecule with a different ssDNA sequence design and/or different fluorophore can be used to form a new DS:IS-fluorophore complex. In one example, the double stranded DNA DS:IS-fluorophore complex can be disrupted with a chemical denaturant, such as concentrated urea, and/or with the application of heat to raise the temperature of the environment above the melting temperature of the complex. After breaking the DS:IS-fluorophore noncovalent bonds, the IS-fluorophore is washed away, and a new IS- fluorophore combination can be added. [0103] Different combinations of IS sequences and conjugated fluorophores are annealed sequentially to the DS (FIGs.1A-1G). A fluorescence lifetime measurement is taken for each IS- fluorophore combination before removing it and annealing a different combination to the DS. In this manner, the NTAA is repeatedly interrogated to yield a characteristic fingerprint of fluorescence lifetime data that differs depending on the identity of the NTAA, the choice of IS sequence, and the choice of fluorophore conjugated to the chosen IS (FIGs.4-9). The library of different IS-fluorophore combinations can be made as broad or as narrow as needed to ascertain the identity of the NTAA as it has been shown that other components of the embodiment do not dominate nor confound the fluorescence (FIG.5). [0104] Next, the N-terminal amino acid is removed from the peptide by performing an Edman degradation. In short, the phenylisothiocyanato (PITC) group of the MPITC linker enables a cyclization reaction with the NTAA that ultimately hydrolyzes the peptide bond between the NTAA and the amino acid in the next position, the “N minus 1” (N-1) position, within the polypeptide chain (FIG. 3). This exposes a new N-terminal amine to which a new MPITC and DS can be Filed: January 4, 2024 conjugated (FIGs.1A-1G). Then, the library of IS-fluorophore combinations is used to interrogate this new NTAA and yield a fingerprint of fluorescence lifetime data based on the identity of this new NTAA (FIGs.7A-7C). [0105] The method described is repeated for each subsequent amino acid until some or all amino acids within the polypeptide chain have been interrogated by the library of IS-fluorophores, and a three-dimension matrix of fluorescence lifetime data for each combination of NTAA, IS sequence, and fluorophore is generated (FIGs.1, 8A-8H, and 10A-10C). The peptide may be up to 50 amino acids in length, for instance, such as 10-50, 10-40, 10-30, 20-50, 30-50, 30-40, 20-40, 20-30, 10- 20, and so forth ranges of amino acids in length. In addition to identification of proteinogenic amino acids, this approach can also be used to detect and identify amino acid post-translational modifications (PTMs) as well as non-canonical or unnatural amino acids (FIGs.6 and 7A & 7B). This matrix of fluorescence lifetime data is then used as an input for a machine learning algorithm, such as a convolution neural network, to ascertain the identity of the amino acids in the peptide chain (FIGs.1 and 10C). The positioning of each amino acid within the peptide chain is known based on the number of Edman degradation cycles performed. For peptides derived from naturally occurring proteins, after identification of some or all of the amino acids in the polypeptide chain the sequence can be aligned to the proteome of the source organism. Within the scientific literature, it has been demonstrated that only a subset of amino acids within an enzymatically digested protein needs to be identified with positional accuracy to ascertain the identity of the protein via proteome alignment (Swaminthan et al., PLOS Comp Biol., 2015; doi.org/10.1371/journal.pcbi.1004080). [0106] In an alternative embodiment (as illustrated in FIG.11), the N-terminus of the peptide is linked to an internally modified nucleotide within the DS oligonucleotide sequence instead of being linked to the terminal end of the DS oligonucleotide. Similarly, the fluorophore is linked to an internally modified nucleotide within the IS oligonucleotide sequence instead of being linked to the terminal end of the IS oligonucleotide. In silico molecular dynamics simulations have shown that for this embodiment, the peptide and fluorophore can be positioned within the major groove of the double stranded DNA to minimize interaction between the fluorophore and the nucleobases of the DS:IS complex. In the other embodiment, the terminal nucleobases of the blunt end of the DS:IS complex might interact with the fluorophore conjugated to the IS and influence the modulation of fluorescence lifetime. Thus, in embodiments, DNA bases or grooves can also interact with dye and/or TAA to confine degrees of freedom or otherwise affect lifetime measurements. Filed: January 4, 2024 (II) Selection & Preparation of Peptides for Analysis [0107] Provided herein are methods for analysis of proteins, polypeptides, and peptides. These methods are applicable to any polypeptide molecules, regardless of their origin. In embodiments, the polypeptides are beneficially processed or prepared in advance of being analyzed. [0108] In some embodiments, the proteins, polypeptides, or peptides to be analyzed are obtained from a biological sample. For instance, a sample may contain mammalian (e.g., human) cells, plant cells, fungal (e.g., yeast) cells, and/or prokaryotic (e.g., bacterial) cells. The sample in some embodiments contains cells that are from a sample obtained from a multicellular organism. For example, the sample may be isolated from an individual (also referred to as a subject, or in some cases a patient). The sample may contain a single cell type or multiple cell types. The sample may include two or more cells. [0109] The sample may be obtained from a mammalian organism or a human, for example by puncture, or other collecting or sampling procedures such as those known in the art. [0110] A peptide may be made of L-amino acids, D-amino acids, or both. A peptide, polypeptide, protein, or protein complex may contain one or more of standard, naturally occurring amino acid(s), modified amino acid(s) (e.g., post-translational modification), amino acid analog(s) or mimetic(s), or any combination thereof. In some embodiments, polypeptide to be analyzed is naturally occurring, synthetically produced, or recombinantly expressed. [0111] Standard, naturally occurring amino acids include Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or Ile), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gln), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). Non-standard amino acids include selenocysteine, pyrrolysine, and N-formylmethionine, β-amino acids, homo-amino acids, Proline and Pyruvic acid derivatives, 3-substituted Alanine derivatives, Glycine derivatives, ring-substituted Phenylalanine and Tyrosine Derivatives, linear core amino acids, and N-methyl amino acids. [0112] In any of the aforementioned embodiments, the peptide, polypeptide, protein, or polypeptide complex may further include one or more post-translational modification(s). A post- translational modification (PTM) of a peptide, polypeptide, or protein may be a covalent modification or enzymatic modification. Examples PTMs include acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, C-terminal amidation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, farnesylation, flavin attachment, formylation, gamma-carboxylation, glutamylation, glycylation, glycosylation (including C-linked, N-linked, O-linked, and phosphoglycosylation), Filed: January 4, 2024 glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenation, succinylation, sulfination, and ubiquitination. [0113] PTMs include modifications of the amino terminus and/or the carboxyl terminus of a peptide, polypeptide, or protein. Modifications of the terminal amino group includes-amino, N- lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A post-translational modification also includes modifications, such those described above, of amino acids falling between the amino and carboxy termini of a peptide, polypeptide, or protein. Post-translational modification can influence the features and/or function(s) of a protein within a cell, e.g., its activity, structure, stability, or localization. For example, phosphorylation plays an important role in regulation of some proteins, particularly in cell signaling (Prabakaran et al., Wiley Interdiscip Rev Syst Biol Med 4: 565-583, 2012). Similarly, the addition of sugars to proteins (e.g., glycosylation) is recognized as promoting protein folding, improving stability, and modifying regulatory function; and attachment of lipids may enable targeting of proteins to the cell membrane. [0114] A post-translational modification can also include peptide, polypeptide, or protein modifications made through experimental or scientific procedures, such as the attachment of detectable label, a linker, and so forth. [0115] Provided herein are methods for assaying (e.g., sequencing) a polypeptide, protein and/or peptide. The methods also permit the analysis of a plurality of different peptides (two or more peptides) simultaneously, e.g., multiplexing. Simultaneously as used herein refers to analysis (for instance, sequencing) of a plurality of peptides with different sequences in the same assay. The plurality of peptides analyzed can be present in the same sample, e.g., biological sample, or different samples. The plurality of polypeptides can be derived from the same subject or different subjects. In some embodiments, the method is performed on a plurality of isolated polypeptides from a sample. In some aspects, the polypeptides are of unknown identity. The plurality of polypeptides that are analyzed can be different polypeptides, or the same polypeptide derived from different samples. A plurality of polypeptides includes 2 or more polypeptides, 5 or more polypeptides, 10 or more polypeptides, 50 or more polypeptides, 100 or more polypeptides, 500 or more polypeptides, 1000 or more polypeptides, 5,000 or more polypeptides, 10,000 or more polypeptides, 50,000 or more polypeptides, 100,000 or more polypeptides, 500,000 or more polypeptides, or 1,000,000 or more polypeptides. Filed: January 4, 2024 [0116] Also contemplated is the simultaneous analysis of different peptide fragments of a single (or plurality of) polypeptide, for instance where the polypeptide(s) is fragmented in some way before the analysis. A plurality of peptide fragments may in various embodiments include 2 or more peptides, 5 or more peptides, 10 or more peptides, 50 or more peptides, 100 or more peptides, 500 or more peptides, 1000 or more peptides, 5,000 or more peptides, 10,000 or more peptides, 50,000 or more peptides, 100,000 or more peptides, 500,000 or more peptides, or 1,000,000 or more peptides. [0117] Thus, in certain embodiments, a peptide, polypeptide, or protein can be fragmented. Peptides, polypeptides, or proteins can be fragmented by any means known in the art, including fragmentation by a protease or endopeptidase, as well as chemical or physical fragmentation. Fragmentation may be carried out through targeted use of a specific protease or endopeptidase that binds and cleaves at a specific consensus sequence. In other embodiments, fragmentation is non-targeted or random by use of a non-specific protease or endopeptidase. A non-specific protease may bind and cleave at a specific amino acid residue rather than a consensus sequence. The following proteinases and endopeptidases, as well as others known in the art (e.g., Granvogl et al., Anal Bioanal Chem 389: 991-1002, 2007), can be used to cleave a protein or polypeptide into peptide fragments: proteinase K (a non-specific serine protease), TEV protease (which cleaves at a specific consensus sequence), trypsin, chymotrypsin, pepsin, thermolysin, thrombin, Factor Xa, furin, endopeptidase, papain, pepsin, subtilisin, elastase, enterokinase, Genenase™ I, Endoproteinase LysC, Endoproteinase AspN, Endoproteinase GluC, and so forth. Also available for use in fragmenting polypeptides before analysis are engineered proteinases, such as thermolabile versions of proteinase K that enable rapid inactivation (see, e.g., WO2019/17089). Proteinase K is also known to be stable in denaturing reagents (such as urea and SDS), which enables digestion of partially or fully denatured proteins. Skilled persons can select a protease from a database based on desired properties of the protease, including specificity to a particular amino acid or sequence of amino acids, known as the protease substrate. Curated proteolytic databases known in the art may include the MEROPS database (accessible at: ebi.ac.uk/merops/), the PANTHER database (accessible at: pantherdb.org), the BRENDA database (accessible at: brenda-enzymes.org), the TopFIND database (accessible at: topfind.clip.msl.ubc.ca), and the UniProt database (accessible at: uniprot.org). [0118] Polypeptides can also fragmented using chemical reagents. Chemical reagents for fragmenting polypeptides or proteins into smaller peptides are known in the art, and include cyanogen bromide (CNBr; which hydrolyzes peptide bonds at the C-terminus of methionine residues), hydroxylamine, hydrazine, formic acid, BNPS-skatole [2-(2-nitrophenylsulfenyl)-3- methylindole], iodosobenzoic acid, NTCB +Ni (2-nitro-5-thiocyanobenzoic acid), and the like. Filed: January 4, 2024 [0119] In certain embodiments, following enzymatic or chemical cleavage, the resulting peptide fragments are approximately the same desired length, e.g., from 10 to 100 amino acids, from 10 to 80 amino acids, from 10 to 60 amino acids, 10 to 40, from 10 to 30 amino acids, from 20 am to 100 amino acids, from 20 to 80 amino acids, from 20 to 60 amino acids, 20 to 40 amino acids, from 20 to 30 amino acids, from 30 to 70 amino acids, from 30 to 60 amino acids, from 30 to 50 amino acids, or from 15 to 40 amino acids. A cleavage reaction may be monitored, for instance in real time, by spiking the protein or polypeptide sample with a short test fluorescence resonance energy transfer (FRET) peptide that contains a proteinase or endopeptidase cleavage site. A fluorescent group and a quencher group are attached to either end of the FRET peptide sequence that includes the cleavage site; FRET between the quencher and the fluorophore leads to low fluorescence. Upon cleavage of the test peptide (for instance, by a protease or endopeptidase) at the included cleavage site, the quencher and fluorophore are separated resulting in a measurable (and quantifiable) increase in fluorescence. This enables stopping the cleavage reaction at a certain fluorescence intensity, which provides a reproducible cleavage endpoint. [0120] In some aspects, the sample can be fractionated, where proteins or peptides are separated by one or more properties (such as cellular location, molecular weight, hydrophobicity, isoelectric point, or protein enrichment methods) in order to reduce the complexity of the sample to be analyzed. Optionally, a subset of macromolecules (e.g., proteins) within a sample is fractionated such that a subset of the macromolecules is sorted from the rest of the sample. The sample may be fractionated prior to attachment to a support. [0121] Alternatively, or additionally, protein enrichment methods may be used to select for a specific protein or peptide (see, e.g., Whiteaker et al., Anal. Biochem. 362:44-54, 2007) or to select for a particular post translational modification (see, e.g., Huang et al., J. Chromatogr. A 1372:1-17, 2014). Alternatively, a particular class or classes of proteins can be affinity enriched or selected for analysis – for instance, by exploiting binding characteristics of such protein class(es). One such class is immunoglobulins, or particular immunoglobulin (Ig) isotypes. In the case of immunoglobulin molecules, analysis of the sequence and abundance or frequency of hypervariable sequences involved in affinity binding are of particular interest, particularly as they vary in response to disease progression or correlate with healthy, immune, and/or or disease phenotypes. [0122] Overly abundant proteins can also be subtracted from the sample using standard methods, including for instance immunoaffinity methods. Depletion of abundant proteins can be useful for plasma samples where over 80% of the protein constituent is albumin and immunoglobulins. Several commercial products are available for depletion of plasma samples of overly abundant Filed: January 4, 2024 proteins, including depletion spin columns that remove top 2-20 plasma proteins (Pierce, Agilent), or PROTIA and PROT20 (Sigma-Aldrich). [0123] A protein, polypeptide, or peptide to be analyzed in accordance with a method described herein may be enriched prior to analysis. Methods for enriching a polypeptide of interest can include removing the polypeptide of interest from a sample (direct or positive enrichment) or removing or subtracting other polypeptides from the sample (indirect or negative enrichment, or depletion), or both. Enrichment can increase the efficiency of the disclosed methods, improve dynamic range, and/or improve the ability to detect low abundance polypeptides in a complex sample. Methods of enrichment can include removing abundant species (that are not an intended target of the analysis), such as albumin; enriching specific targeting of particular proteins (e.g., by antibody or other affinity capture) (or subtracting non-targets through such capture); enriching using one or more general properties of proteins (e.g., size, pI, hydrophobicity, etc.) (or subtracting non-targets using those properties); enriching by targeting classes of polypeptides (e.g., by post-translational modification(s), such as phosphorylated proteins and glycosylated proteins) (or subtracting non-targets); by ability to bind certain molecules (e.g., DNA binding proteins); ATP binding proteins; enrich/subtract by subcellular localization (e.g., nuclear, mitochondrial, Golgi, endoplasmic reticulum, and so forth); enriching by the cellular population (e.g., T-cells, B-cells, etc.) that produces the target polypeptide, where the cell can be identified, sorted or otherwise captured (e.g., via cell surface markers). Art-recognized methods and techniques for enrichment include centrifugation, chromatography, electrophoresis, binding, filtration, precipitation, and degradation. The dynamic range of a sample also can be modulated by fractionating the sample using standard fractionation methods, such as electrophoresis and liquid chromatography (Zhou et al., Anal Chem 84(2): 720-734, 2012). (III) Blocking Undesirable Chemical Reactivity with Protecting Groups [0124] Before or after enzymatic or chemical digestion of a polypeptide, reactive side chains or chemical groups can be blocked with protecting groups to prevent side reactions, for instance in the subsequent conjugation reactions. Such reactive groups include amino, carboxyl, sulfhydryl, hydroxyl, and guanidino side chains. A protecting group (PG) is a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in that reactive portion of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group may be removed under conditions that do not degrade or decompose the remaining portions of the molecule, i.e. the protected reactive portion of the molecule is “deprotected”. Protecting groups (PGs) and their reactions are well-understood and may be chosen by a person skilled in the art based on compatibility with downstream chemical Filed: January 4, 2024 reactions (Isidro-Llobet et al., Chem Ref. 109(6):2455-2504, 2019; doi.org/10.1021/cr800323s; Protecting Groups 3^rd Ed. ISBN-13: 978-1588902351). Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973, in Greene, T. W. and Wuts, P. G. M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3rd Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of protecting groups include t-Boc, C1-6acyl, Ac, Ts, Ms, silyl ethers such as TMS, TBDMS, TBDPS, Tf, Ns, Bn, Fmoc, dimethoxytrityl, methoxyethoxymethyl ether, methoxymethyl ether, pivaloyl, p-methyoxybenzyl ether, tetrahydropyranyl, trityl, ethoxyethyl ethers, carbobenzyloxy, benzoyl, and the like. For instance, the protecting group in some instances is an amine protecting group. [0125] Protecting groups can be added either before enzymatic cleavage (which might require sacrificing the N- and C-terminal component peptides as the N-terminal amine and C-terminal carboxylic acid of the protein might also be protected) – this would render them inert to the downstream reactions of a provided sequencing protocol. Protecting groups can be added after enzymatic digestion, which employs use of specific protecting agents that would not protect the N- or C-terminal functional groups of the component peptides. Examples of such protecting groups are known in the art, and are exemplified herein. [0126] By way of example, attachment of a docking strand to NTAA in embodiments is via the terminal amine and an isothiocyanate (ITC) group. ITCs may react with alpha amine on NTAA, as well as epsilon amino on Lysine, and to a lesser extent thiol on cysteine. These residues within peptides to be analyzed may be capped/protected. For amines, the NTAA and lysine amines may be capped/protected, and then the NTAA cap can be removed by Edman degradation to reveal a fresh amine. The lysine(s) remain capped because the amide bond is not cleaved under Edman conditions. [0127] Advantageously, protecting groups can be selected/designed to have altered interactions between the fluorophore and the protected sidechain compared to the unprotected sidechain when it is interrogated as the TAA. This could have a significant effect on lifetime changes, which can be taken into account in processing signals used to train a database and therefore to identify amino acids in test analyses. Thus, embodiments are contemplated in which analysis of target polypeptides includes analysis with differently modified (e.g., protected) sidechain(s) and comparison of the resultant changes in spectral analyses. (IV) Support Surface for Immobilization of Peptides [0128] In described polypeptide analysis methods, the polypeptides, proteins, or peptides are immobilized on a surface (a support surface) by one terminus (either the amino- or carboxy- Filed: January 4, 2024 terminus), and the peptides (e.g., all of the peptides) in any “run” are attached by the same end. Attachment to a support surface enables reliable interrogation of each individual feature (that is, the location at which each peptide is immobilized), including through multiple steps or cycles of analysis. [0129] The polypeptides, proteins, or peptides can be joined to (covalently attached to) a support surface, directly or indirectly, by any means known in the art. In some cases, it is desirable to use a support with a large carrying capacity to immobilize a large number of (different) polypeptides. [0130] Solid support surfaces to which proteins, peptides, and polypeptides can be attached are known in the art. See, for instance, descriptions provided in US Patents No. 7,972,827, 10,852,305, 11,105,812, and 11,268,963; as well as published patent applications US 2022/0155316, US 2021/0396762, WO 2010/065531, and WO 2016/069124. [0131] Support surfaces may include any substrate (such as glass, quartz, plastic, silicon, silicon oxide, ceramic, metals, metal oxides, alloys, or semiconductors) of any dimensions on which a biological sample (e.g., containing polypeptides or peptides) is placed (or arrayed) for analysis (thus, also an “analysis substrate”). In embodiments, the support surface may be a microscope slide such as a standard 3'' x 1'' glass slide or a standard 75 mm x 25 mm glass slide. Additional examples of substrates include substrates, such as mass spectrometry platforms, used to assist in analysis of a sample, such as SELDI and MALDI chips. The proteins, peptides, and polypeptides described herein can be applied to whatever type of analysis substrate is typically used for detection of the type of signal that is being employed. In some embodiments, the support is a planar substrate. [0132] In some embodiments, polypeptides are immobilized using a three-dimensional support (e.g., a porous matrix or a bead). In other embodiments, polypeptides are immobilized using a support compatible with the signal detection method, sensor, and/or device that will be used in the analysis. Solid or semi-solid supports can include surfaces, such as glass, plastic, ceramic, and/or metal; particles, such as nanometer-, micrometer-, or millimeter-sized particles composed of materials including polystyrene, iron oxide, tentagel, glass, ceramics, and/or plastic; and other shapes and forms of matter. In certain embodiments, a support is a bead (or collection of beads), for example, a polystyrene bead, a polymer bead, a polyacrylate bead, an agarose bead, a cellulose bead, a dextran bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a silica-based bead, or a controlled pore bead, or any combinations thereof. In embodiments, the support is a bead array. While it is contemplated that beads may be used as the support surface, porous beads may allow disadvantageous inter- peptide crosstalk. Loading density can be optimized to minimize this issue. Filed: January 4, 2024 (V) Support Surface Conjugation of Peptides [0133] Various reactions may be used to attach the polypeptides to a support surface (e.g., a solid or a porous support). The polypeptides may be attached directly or indirectly (for instance, via a linker) to the support. [0134] Various methods for attaching proteins to solid support surfaces are known in the art. See, for instance, Chan et al. (PloS One, 2(11):e1164, 2007, doi: 10.1371/journal.pone.001165), Camarero & Kwon (Int J Peptide Res Therap.14:351-357, 2008); Camarero et al. (J Am Chem Soc.126(45):14730-14731, 2024); Kwon et al. (Angewandte Chemie, 45(11):1725-1729, 2006, doi.org/10.1002/anie.200503475); US Patents No. 7,972,827, 10,852,305, 11,105,812, and 11,268,963; and published patent applications US 2022/0155316, US 2021/0396762, WO 2010/065531, and WO 2016/069124. [0135] Exemplary reactions include the copper catalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1, 3-dipolar cycloaddition), strain-promoted azide alkyne cycloaddition (SPAAC), reaction of a diene and dienophile (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reaction of a strained alkene with an azide, tetrazine or tetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse electron demand Diels-Alder (IEDDA) reaction (e.g., m-tetrazine (mTet) or phenyl tetrazine (pTet) and trans-cyclooctene (TCO)); or pTet and an alkene), alkene and tetrazole photoreaction, Staudinger ligation of azides and phosphines, and various displacement reactions, such as displacement of a leaving group by nucleophilic attack on an electrophilic atom (Horisawa, Front Physiol.5:457, 2014, doi: 10.3389/fphys.2014.00457; Knall et al., Tetrahedron Lett.55(34):4763-47662014, doi: 10.1016/j.tetlet.2014.07.002). [0136] Exemplary displacement reactions include reaction of an amine with: an activated ester; an N-hydroxysuccinimide ester; an isocyanate; an isothiocyanate, an aldehyde, an epoxide, or the like. In some embodiments, iEDDA click chemistry is used for immobilizing polypeptides to a support since it is rapid and delivers high yields at low input concentrations. In another embodiment, m-tetrazine rather than tetrazine is used in an iEDDA click chemistry reaction, as m-tetrazine has improved bond stability. In another embodiment, phenyl tetrazine (pTet) is used in an iEDDA click chemistry reaction. In one case, a polypeptide is labeled with a bifunctional click chemistry reagent, such as alkyne-NHS ester (acetylene-PEG-NETS ester) reagent or alkyne- benzophenone to generate an alkyne-labeled polypeptide. In some embodiments, an alkyne can also be a strained alkyne, such as cyclooctynes including Dibenzocyclooctyl (DBCO), and so forth. [0137] To interrogate multiple peptides on an immobilization surface, the minimum distance between peptides in some embodiments is 2 nanometers, or more. The minimum distance may be influenced by resolution of the optical instrument reading the fluorescence lifetimes (or other Filed: January 4, 2024 signal from the IS, and which is influenced by the local molecular environment). Methods for measuring the required minimum, or optimized, or optimum, distance between peptides on an immobilization surface in order to enable optical detection of individual features, and how to separate readings from those features (e.g., assisted by computer analysis of the signals), are well known in the art. [0138] Spacing will be particularly limited by fluorescence detection resolution. On a widefield or confocal microscope, resolution is limited by diffraction of the photon. This is proportional to the wavelength (lambda) of the photon, and numerical aperture of the objective (NA): Resolution ≈ (lambda)/(2xNA). When using super resolution approaches such as DNA-PAINT or dSTORM, this resolution can vary but typically does not get better than 10 nanometers. This is the smallest separation that can be achieved between peptides on examples of the array. This is also on the same order of magnitude as the Förster resonance energy transfer (FRET) effect, where donor and acceptor fluorophores or quenchers tend to interact when spacing between them is about 10 nanometers or less. The contribution of an amino acid side chain on an adjacent molecule may contribute to energy transfer (such as quenching or FRET) at spacing below 10 nanometers. However, deconvolution or denoising of the proximal (desired amino acid) and distal (adjacent amino acid crosstalk) effects of amino acids on the fluorescence lifetime may be possible if distal contributions are sufficiently small (empirical evidence doesn’t exist). In this case, peptide/oligonucleotide barcoding, substrate patterning or sparse/stochastic labeling and/or fluorophore emission may support spacing well below 10 nanometers. Both of these spacing limits could theoretically be solved by using nanometer sized wells or pits with radii less than 5 nm, that only a single complex could occupy. In this case, the physical boundaries of the well could inhibit crosstalk, and a single-sensor based detection at the base of each pit or well could solve the diffraction-limited resolution issue. Might also include nanoneedles or DNA origami. These may provide sub 10 nm spacing as well and can be mixed with discrete detection sensors similar to Pac Bio. Another possibility is to use an AFM cantilever to scan samples or attach the molecules to an array of hollow nano pyramids/pillars and use a near-field fluorescence approach. For AFM NSOM throughput may be low and may relegate spacing to approximately 50 nm. See also the teachings in Pan et al. (Optics Comm. 445:273-276, 2019, doi.org/10.1016/ j.optcom.2019.04.053) as well as the description of Near-Field Scanning Optical Microscopy provided by Olympus Lifesciences (available online at olympus-lifescience.com/en/microscope- resource/primer/techniques/nearfield/nearfieldintro/). [0139] Thus, in certain embodiments where multiple polypeptides are immobilized on the same support, the polypeptides can be spaced appropriately to accommodate methods of performing the binding reaction and any downstream detection and/or analysis steps to be used to assess Filed: January 4, 2024 the polypeptide. For example, it may be advantageous to space the molecules optimally for the signal detection step. In some cases, the appropriate spacing depends on the type of signal generated and detection method or sensor used to detect the signal. In some cases, spacing of the targets on the support is determined based on the consideration that a signal generated in association with one polypeptide may obscure or be indistinguishable with a signal generated with a neighboring molecule. In some embodiments, the polypeptides are immobilized on a support and spaced at optically resolvable distances. [0140] In some embodiments, the surface of the support is blocked - a surface that has been treated with a layer of material. Methods of blocking surfaces include standard methods that were originally developed for fluorescent single molecule analyses, including blocking surfaces with polymer like polyethylene glycol (PEG) (Pan et al., Phys. Biol. 12:045006, 2015), polysiloxane (e.g., Pluronic F-127), star polymers (e.g., star PEG) (Groll et al., Methods Enzymol.472:1-18, 2010), hydrophobic dichlorodimethylsilane (DDS)+self-assembled Tween-20 (Hua et al., Nat. Methods 11:1233-1236, 2014), diamond-like carbon (DLC), DLC+PEG (Stavis et al., Proc. Natl. Acad. Sci. USA 108:983-988, 2011), and zwitterionic moiety (e.g., US 2006/0183863). In addition to covalent surface modifications, a number of blocking agents can be employed - including surfactants like Tween-20, polysiloxane in solution (Pluronic series), poly vinyl alcohol (PVA), and proteins like BSA and casein. [0141] In embodiments, the density of proteins, polypeptide, or peptides can be titrated on the surface or within the volume of a solid substrate by spiking a competitor or “dummy” reactive molecule when immobilizing the proteins, polypeptides, or peptides to the solid substrate. [0142] Spacing of the immobilized polypeptides on the support can also be controlled by modifying (titrating) the density of functional coupling groups for attaching the polypeptides (e.g., TCO or carboxyl groups (COOH)) on the substrate surface. In some embodiments, multiple molecules are spaced apart on the surface or within the volume (e.g., porous supports) of a support such that adjacent molecules are spaced apart at a distance of 50 nm to 500 nm, or 50 nm to 400 nm, or 50 nm to 300 nm, or 50 nm to 200 nm, or 50 nm to 100 nm. In some embodiments, multiple molecules are spaced apart on the surface of a support with an average distance of at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, or at least 500 nm. [0143] Thus, in some embodiments, appropriate spacing of the on the support is accomplished by titrating the ratio of available attachment molecules on the substrate surface. In some examples, the substrate surface (e.g., bead surface) is functionalized with a carboxyl group (COOH) that is treated with an activating agent (e.g., EDC and Sulfo-NHS). In some examples, Filed: January 4, 2024 the substrate surface (e.g., bead surface) includes NHS moieties. In some embodiments, a mixture of mPEG_n-NH₂ and NH₂-PEG_n-mTet is added to the activated beads (wherein n is any number, such as 1-100). The ratio between the mPEG₃-NH₂ (not available for coupling) and NH₂- PEG₂₄-mTet (available for coupling) may be titrated to generate an appropriate density of functional moieties available to attach the polypeptides on the substrate surface. In certain embodiments, the mean spacing between coupling moieties (e.g., NH₂-PEG₄-mTet) on the solid surface is at least 50 nm, at least 100 nm, at least 250 nm, or at least 500 nm. In some embodiments, the spacing of the polypeptides on the support is achieved by controlling the concentration and/or number of available COOH or other functional groups on the support. (VI) Modification of Free End of Immobilized Peptides [0144] Optionally, in some embodiments the terminal amino acid of the polypeptide may be derivatized prior to conjugating a Docking Strand (DS) to the terminal amino acid, in order to enable or enable that conjugation. For example, in one embodiment the terminal amino acid is a NTAA, and that NTAA is derivatized with an Edman reagent such as phenyl isothiocyanate (PITC). [0145] Also contemplated are embodiments in which an isoselenocyanate group is used in place of isothiocyanate; this may be substituted to provide schemes analogous to those provided herein. [0146] The herein described and produced 1-(4-isothiocyanatophenyl)-1H-pyrrole-2,5-dione (CAS Reg. No.60283-89-8, also referred to as maleimidophenyl isothiocyanate or MPITC; Keana et al., J Am Chem Soc.108:7947-7963, 1986; available commercially from Chemieliva Biotech Co. Limited, Chongqing, China) provides beneficial properties in allowing both docking strand conjugation and Edman degradation capabilities (via the maleimide and isothiocyanate groups, respectively). Other bifunctional linkers were capable of only one of these functions, limiting their usefulness and requiring multiple linkers to replicate MPITC’s functionality. [0147] The final product was obtained from a commercially available precursor 1-(4- aminophenyl)-1H-pyrrole-2,5-dione (CAS Reg. No. 29753-26-2). The synthesis foresees the creation of an intermediate by reacting the precursor’s aniline group with carbon disulfide in the presence of DMAP and ET3N. The reaction was completed by reacting the highly energetic intermediate with Boc₂O under anhydrous conditions. Filed: January 4, 2024 [0148] A representative reaction scheme is as follows: [0149] The penultimate intermediate compound in the scheme above may be referred to as (4- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)carbamothioic pivalic thioanhydride. Additional intermediate compounds for use in this process include, but are not limited to, (4-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,6-dimethylphenyl)carbamothioic pivalic thioanhydride; (4-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)-2,6-dihydroxyphenyl)carbamothioic pivalic thioanhydride; and (4-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,6-dimethoxyphenyl)carbamothioic pivalic thioanhydride. Filed: January 4, 2024 [0150] The central phenyl structure provides rigidity, and therefore fewer degrees of freedom between the conjugated modules ensuring consistent interactions, a useful feature for kinetic- sensitive signaling, such as fluorescence, chemiluminescence, etc. [0151] The maleimide allows for conjugation with a range of thiol and amine-containing molecules, expanding its use to outside of single-type systems to include other potential biological markers, such as antibodies, modified oligonucleotides, varying peptides, proteins, etc. At the same time, the mildness of the synthesis allows for other coupling groups to be attached to the linker, including DBCO, NHS ester, TCO, azide, etc. without disturbing the ITC formation. [0152] The MPITC linker optionally can be improved for FLIM use by adding electron donating groups on ortho position to the ITC. The resulting enhanced electron density can potentially make Edman Degradation faster, improving the efficiency of the workflow. A scheme with a few examples is as follows: b) 1-(4-isothiocyanato-3,5-dimethylphenyl)-1H-pyrrole-2,5-dione; c) 1-(3,5-dihydroxy-4-isothiocyanatophenyl)-1H-pyrrole-2,5-dione; and d) 1-(4-isothiocyanato-3,5-dimethoxyphenyl)-1H-pyrrole-2,5-dione. [0153] In further embodiments, the peptide analyses (including polypeptide sequencing) described herein can be performed with C-terminal attachment of the oligonucleotide docking strand (DS) and amino acid removal via C-terminal degradation. First, the N-terminus of the peptide is attached to a substrate using standard amine coupling reagents/protocols. [0154] A C-terminal thiohydantoin moiety is formed and then, a DS bearing a leaving group, including but not limited to acyl halides (e.g. acyl chloride, acyl bromide, acyl iodide, etc.) and tosylates, is used to S-alkylate the thiohydantoin. This alkylation constitutes the addition of a DS Filed: January 4, 2024 to the C-terminus, which enables application of the FLIM workflow via sequential imaging strand (IS)-fluorophore interrogation of the C-terminal amino acid (CTAA). Interrogation of the CTAA side chain is performed in the same way as interrogation of the NTAA with an N-terminally-attached DS. An IS-fluorophore conjugate is annealed to bring the fluorophore into proximity of the CTAA or “C–1” amino acid side chain, which is determined according to linker composition and length; the fluorescence lifetime of the fluorophore is measured; the IS-fluorophore is removed via heating, chemical denaturation, or a combination of both; another IS-fluorophore for which the IS, fluorophore, or both are different is annealed; and the interrogation workflow is cycled until a sufficient amount of modulated fluorescence lifetime data is acquired for the CTAA or “C-1” amino acid. [0155] To remove the CTAA and to form C-terminus from the “C–1” position amino acid, Schlack- Kumpf degradation is performed (scheme provided below; see also Li & Liang, Anal Biochem 302(1):108-0113, doi.org/10.1006/abio.2001.5505). This cleavage reaction reforms the peptide- thiohydantoin at the C-terminus of the N-terminally immobilized peptide. After cleavage of the original “C” position amino acid CTAA from the peptide, the “C–1” position amino acid constitutes a newly-formed C-terminus and can then be referred to as the CTAA. [0156] In this method, the C-terminus is first activated and converted to a thiohydantoin moiety. The C-terminal carboxylic acid is converted to an anhydride group by combining with acetic anhydride for 5 min at 50 to 80 ºC before addition of 0.5 M triphenylgermanyl isothiocyanate (Ph3Ge-ITC), or a similar, highly substituted analog, in acetonitrile. The peptide-thiohydantoin is alkalized with reagents that can include triethylamine, sodium bicarbonate, and sodium borate and then combined with oligonucleotide docking strand (DS) modified with a leaving group, such as acyl chloride or others. The leaving group can be positioned at the 5’ end, 3’ end, or within the DS depending on which interrogation scheme will be used. This results in conjugation of the DS to the C-terminus of the peptide via a thiolate moiety (Boyd et al., Anal Biochem.206(2):344-352, 1992, doi.org/10.1016/0003-2697(92)90376-i). [0157] After fluorescence lifetime data is acquired, C-terminal degradation is performed via addition of hydrogen isothiocyanate (or isothiocyanate anions), which can be generated from donors including (trimethylsilyl)isothiocyanate, under acidic conditions. This results in cleavage of the CTAA-DS conjugate from the peptide. This cleavage reaction reforms the thiohydantoin at the C-terminus of the peptide from the original “C–1”-position amino acid (see scheme below). Filed: January 4, 2024 (DS) via the thiolate moiety and subsequent C-terminal degradation and removal of the CTAA. DS attachment to the peptide can be at the 3’ end, 5’ end, or internally. Upon cleavage of the CTAA, a thiohydantoin moiety is reformed, and the DS can be attached to the newly formed C- terminus via an alkylation reaction without the need for reactivation of the C-terminus. Not shown in this schematic is the substrate attachment of the peptide via its N-terminal amine or the repeated interrogation (via IS-fluorophore annealing and fluorescence lifetime data acquisition). [0159] While methods are exemplified herein using isothiocyanate (ITC, R-N=C=S), ITC analogs, such as isoselenocyanate (ISC, R-N=C=Se), are also contemplated. Thus, variations of the peptide-to-docking strand linker are contemplated to include an ISC group in place of the ITC, and that the Edman degradation could be performed by an ISC instead of an ITC. [0160] In embodiments of the peptide analysis/sequencing workflow (exemplified by FLIM), including those using N-terminal (or C-terminal) degradation approaches, isothiocyanate analogs can instead be used. These analogs include isoselenocyanates (ISC) (Maeda et al., Heterocycles 82(2):2010 doi.org/10.3987/com-10-s(e)116; Iskierko et al., Ann Univ Mariae Curie Sklodowska Med (in pol), 3169-76, 1976). It can be beneficial to use such analogs as they exhibit varying levels of reactivity and, thus, can enable design variations in the different reaction conditions used throughout the FLIM workflow. This facilitates the use of milder conditions, which can include shorter reaction times, lower temperatures, and less extreme pH. Milder conditions can enhance Filed: January 4, 2024 the relative stability of the structures (peptide, etc.) and linkages (peptide-to-surface, etc.) used within the workflow, which can enhance data fidelity. [0161] Also provided herein are compounds of Formula (I): or a salt, or solvate thereof, x is 0, 1 or 2; each R independently is selected from the group consisting of C₁-C₆ alkyl, -NO₂, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO₃, or any other common electron withdrawing group; R¹ and R² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, -O-(C₁-C₆ alkyl), C1-C6 alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)- R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, halogens, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N- (C=O)-OR, or any other common electron donating group.

Filed: January 4, 2024 [0162] Also provided are compounds of Formula (II): or a salt, or solvate x is 0, 1 or 2; each R independently is selected from the group consisting of C₁-C₆ alkyl, -NO₂, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO₃, or any other common electron withdrawing groups; R¹ and R² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, -O-(C₁-C₆ alkyl), C₁-C₆ alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)- R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N- (C=O)-OR, or any other common electron donating groups. [0163] Also contemplated are compounds of Formula (I) or Formula (II), wherein the six- membered ring (illustrated below) could be replaced by other aromatic groups (e.g., naphthyl, other benzo fused rings), heteroaryl, heterocyclyl, or alkyl groups, each of which may be unsubstituted or substituted. Filed: January 4, 2024 (VII) Signal Molecules [0164] In the provided polypeptide analysis methods, individual terminal amino acids are distinguished based on their impact on the (molecular-scale) local environment. That impact is detected, and distinguished between, by using signal compound(s)/molecules that exhibit a detectable/measurable change in a measurable characteristic, based on that impact. By way of example, the signal compounds have one or more spectral properties that are influenced by the proximity of one or more amino acid side groups – and that influence is detected by analyzing and comparing the spectral property(s). [0165] In one embodiment, the protein analysis methods include comparing one or more spectral properties of a signal molecule in proximity to a terminal amino acid (e.g., a NTAA or CTAA) of an immobilized polypeptide to a set of reference spectral properties of the interaction of the signal molecule when it is in proximity to a known terminal amino acid. In one embodiment, the spectral property includes a fluorescence signal from a fluorophore, luminescence from a luminescent molecule, phosphorescence from a phosphorescent molecule. Luminescent molecules, which do not require heat (i.e. photons) to be excited, as well as phosphorescent molecules typically have longer lifetimes than fluorescence (1 µs to several seconds). With current opto-electronics, FLIM instrument sensitivity is about 0.05-0.3 ns, which limits detection of small differences in amino acids in this embodiment. The use of molecules that possess longer lifetimes may increase sensitivity; however, this may increase the overall measurement time. [0166] In one embodiment, appropriate signal compounds exhibit different spectral properties when near (proximal to) different N-terminal amino acids. For example, various fluorescent dyes are shown herein to exhibit predictable and variable spectral properties (typified herein with fluorescence lifetime) when in proximity to different amino acid residues. [0167] By way of specific examples, the following fluorophores (each of which is commercially available) are shown herein to be able to “detect” differences in proximal amino acids, based on changes in their fluorescence lifetime as measured by FLIM.

Filed: January 4, 2024 [0168] AF488 (Lifetime = 4.1 ns) [0169] BODIPY FL (Lifetime = 5.87 ns)

Filed: January 4, 2024 [0170] BODIPY TR (Lifetime = 5.7 ns) [0171] TAMRA (Lifetime = 5.7 ns)

Filed: January 4, 2024 [0172] KU530-6 (Lifetime = ~24 ns) [0173] KU530-R-4 (Lifetime = ~24 ns)

Filed: January 4, 2024 [0174] KU560-6 (Lifetime = ~20 ns) [0175] [0176] Thus, signal molecules can be a fluorescent dye, and the spectral characteristic may be the lifetime of fluorescence from that dye and how it changes when in proximity of different peptide-terminal amino acids. Fluorescent dyes can be detected in real time with high resolution, and many fluorescent dyes are available hat have distinct excitation and emission wavelengths. Sets of fluorescent dyes can be selected so as to allow for a simultaneous detection of more than one dye in the same reaction, for instance as a way to control of complete washing (removal) of a prior interrogation IS. The following dyes, for instance, can be detected at the same time and Filed: January 4, 2024 distinguished: Cy3, Cy5, FAM, JOE, TAMRA, ROX, dR110, dR6G, dTAMRA, and dRox. Any of those dyes can be used individually or in any combination to practice an embodiment herein. [0177] A dye can allow for single molecule detection. A large number of fluorescent dyes have been synthesized, and are commercially available in different formats (see, for instance, compounds available from Invitrogen). This can include fluorescent dyes having a linker region and a hydrazine group that allows for coupling to a nucleic acid in a reaction with dialdehyde groups. [0178] In some embodiments, 2-3 fluorophores are conjugated sequentially to the 5’ end of the IS and act a FRET pairs. Being of a set rigid distance between each donor and acceptor (10 angstroms-10 nm), more than 1 amino acid residue along the peptide sequence is interrogated and fluorescence lifetimes are collected from all fluorophores. When performing FLIM or another peptide sequencing method provided herein, the lifetime can change based on proximal amino acid as well as nearby FRET donor/acceptor, thus providing more information about the environment. [0179] The present disclosure is not limited to the use of a specific fluorescent dye, but different dyes can be applied to the same effect. In fact, libraries of two or more different fluorescent molecules are demonstrated herein to provide better characterization of amino acids. This is demonstrated herein by sequential interrogation of the same terminal amino acid with more than one signal molecule (e.g., more than one different fluorophore), each attached do an IS that is bound serially to the DS attached to immobilized peptides. The different readouts form each interaction can be used to increase the fidelity of amino acid identification. [0180] Non-limiting examples of signal molecules can include 5-FAM (also called 5- carboxyfluorescein; 6-Carboxy-4',5'-dimethylfluorescein (also called Spiro[isobenzofuran-1(3H), 9'-(9H)xanthene]-5-carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein; Cdmfda); 5- Hexachloro-Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro-Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5- carboxylic acid]); 5-Tetrachloro-Fluorescein; ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)- 5-carboxylic acid]); 6-Tetrachloro-Fluorescein; ([4,7,2',7'-tetrachloro-(3',6'-dipivaloyl- fluoresceinyl)-6-carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine); Xanthylium, 9- (2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine); 9- (2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1- sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5 (Indodicarbocyanine-5); Cy3 (Indo-dicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionic acid); Quasar™-670 dye (Biosearch Technologies); Cal Fluor™ Orange dye (Biosearch Technologies); Rox dyes; Max Filed: January 4, 2024 dyes (Integrated DNA Technologies), tetrachlorofluorescein (TET), 4,7,2'-trichloro-7'-phenyl-6- carboxyfluorescein (VIC), HEX, Cy3, Cy 3.5, Cy 5, Cy 5.5, Cy 7, tetramethylrhodamine, ROX, and JOE as well as suitable derivatives thereof. The label can be an Alexa Fluor® dye, such as Alexa Fluor® 350, 405, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, and 750. The label can be Cascade Blue, Marina Blue, Oregon Green 500, Oregon Green 514, Oregon Green 488, Oregon Green 488-X, Pacific Blue, Rhodamine Green, Rhodol Green, Rhodamine Green-X, Rhodamine Red-X, and Texas Red-X. The label can be a set of longer lifetime dyes, such as from the KU™ dye family (KU450, KU470, KU483, KU500, KU510, KU530, KU542, KU560, KU600, KU600, KU625, KU-P, and KU-T) (KU Dyes, Department of Chemistry, University of Copenhagen, Denmark). The label can be at the 5' end of a probe, 3' end of the probe, at both the 5' and 3' end of a probe, or internal to the probe. A distinguishable (e.g., unique) label can be used to detect each different locus in an experiment, for example two termini of a target polynucleotide, such as mRNA. [0181] Non-limiting examples of dye-hydrazides that can be used as signal molecules include Alexa Fluor™-hydrazides and salts thereof, 1-pyrenebutanoic acid-hydrazide, 7- diethylaminocoumarin-3-carboxylic acid-hydrazide (DCCH) Cascade Blue™ hydrazides and salts thereof, biocytin-hydrazide, 2-acetamido-4-mercaptobutanoic acid-hydrazide (AMBH), BODIPY™ FL-hydrazide, biotin-hydrazide, Texas Red™-hydrazide, biocytin-hydrazide, luminol (3-aminophthalhydrazide), and Marina Blue™ hydrazide. Non-limiting examples of dye- ethylenediamines that can be used for labeling include 5-dimethylaminonaphthalene-1-(N-(2- aminoethyl))sulfonamide (dansyl ethylenediamine), Cascade Blue™ ethylenediamine and salts thereof, N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalimide (lucifer yellow ethylenediamine) and salts thereof, N-(biotinoyl)-N'-(iodoacetyl) ethylenediamine, N-(2-aminoethyl)biotinamide, hydrobromide (biotin ethylenediamine), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-propionyl ethylenediamine and salts thereof (BODIPY™ FL EDA), Lissamine.TM. rhodamine B ethylenediamine, and DSB-X.TM. biotin ethylenediamine (desthiobiotin-X ethylenediamine, hydrochloride). [0182] Non-limiting examples of dye-cadaverines that can be used as signal molecules include 5-dimethylaminonaphthalene-1-(N-(5-aminopentyl))sulfonamide (dansyl cadaverine), 5-(and-6)- ((N-(5 aminopentyl) amino) carbonyl) tetramethylrhodamine (tetramethylrhodamine cadaverine), N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide and salts thereof (lucifer yellow cadaverine), N-(5-aminopentyl)biotinamide and salts thereof (biotin cadaverine), biotin-X cadaverine (5-(((N-(biotinoyl) amino) hexanoyl) amino) pentylamine and salts thereof, Texas Red™ cadaverine (Texas Red™ C5), 5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s- indacene-3-yl) phenoxy) acetyl) amino) pentylamine and salts thereof (BODIPY™ TR Filed: January 4, 2024 cadaverine), Oregon Green™ cadaverine, Alexa Fluor® cadaverine, and 5-((5-aminopentyl) thioureidyl) fluorescein and salts thereof (fluorescein cadaverine). [0183] Alternative spectroscopic techniques such as anisotropy or fluorescence polarization, may be used to enhance distinguishing amino acid residues along a peptide. An amino acid in close proximity to a fluorophore may alter the spatial orientation of the fluorophore on the IS. This may affect the direction of emission from being uniform, isotropic, to directionally dependent, or anisotropic. These spectroscopic techniques can be combined with the existing FLIM technique discussed in this art by polarizing the excitation light and increasing the number of detectors within the current optical path. Therefore, by collecting spatial and temporal information from fluorophores (i.e., anisotropy and fluorescence lifetime, respectively), the sensitivity to determine differences in amino acids may increase, especially when determining post-translational modifications. (VIII) Imaging Strands, and Attachment of Signal Molecules [0184] A ssDNA imaging strand (IS) with an attached signal molecule is used in the provided peptide sequencing methods, in which the IS is annealed to a single stranded docking stand (DS) to form a double stranded DS:IS complex. The IS bears one or more signal molecules, which is/are attached covalently to the IS oligonucleotide at or near one end or the other, or internally, depending on the embodiment. [0185] The designed ssDNA IS of a chosen sequence with a signal label attachment modification, for instance on the 5’ end of the oligonucleotide. The signal label attachment modification is tailored based on the type of signal molecule that will be attached to the IS, to enable the attachment chemistry. In one example, this modification is an alkyl amine such as (6- aminohexyl)phosphate, which is conjugated covalently to the maleimido group of a maleimide- functionalized fluorophore; this is illustrated in FIGs.2 and 3 for instance. [0186] The length of the IS may be influenced by the melting temperature of the DS:IS complex. Designing IS with certain guanine and cytosine content and adjusting environmental salt conditions can assist in producing the desired IS. The minimal length of IS in embodiments is 8- 10 bases. The maximum length of IS may be influenced (e.g., constrained) by the upper range of the melting temperature of the DS:IS complex. DNA hybridization melting temperature (T_m) is affected by salt concentration in the reaction buffers and washes. If employing PAINT techniques then lower salt, higher temperature, and/or co-solvents can tune those kinetics. Similarly, dyes that have low aqueous solubility could benefit from having some solvent (DMSO, DMF, PEG) Washes to remove imaging strands can be improved through use of detergents (anionic, zwitterionic, cationic). Filed: January 4, 2024 [0187] In FLIM, many different dyes (signal molecules) can be used that have different spectral properties (excitation/emission wavelengths, lifetimes, and so forth). Lifetime of each dye may be affected differently depending on the TAA (e.g., NTAA) in proximity to it. Thus, a set of dyes could provide a unique signal for each NTAA. Better sensitivity and resolution achieved by restricting the dyes movement or degrees of freedom about the NTAA. Attaching the docking strand using internal modified bases and using Imaging strands with dye attached through internal base modifications can help position the dye and NTAA along major or minor groove of DNA. This could result in more reproducible or consistent lifetime effects [0188] Molecular dynamics simulations were performed to model the interactions of the fluorophore of the DNA imaging strand (IS)-fluorophore conjugate with the side chain of the N- terminal amino acid (NTAA). The lengths and chemical composition of the various linkers involved in the workflow (FIG. 3) were optimized based on the propensity for the NTAA side chain to interact with the fluorophore. Similarly, variations in the IS sequence, which can result in overhangs or underhangs relative to the DNA docking strand (DS) were also modelled (FIG.2). For certain NTAAs, the change in the fluorescence lifetime of a particular fluorophore could be predictively modelled in silico based on the proportion of time in which the side chain of the amino acid interacts with the fluorophore. For example, Alexa Fluor® 488 (AF488) interacts with the side chain of an N-terminal tryptophan group for a greater portion of a simulation than do arginine or glycine, so a person skilled in the art can predict that for the modelled combination of IS and fluorophore, that an N-terminal tryptophan residue is expected to suppress the fluorescence lifetime of AF488 more than an N-terminal arginine or glycine residue. [0189] Also contemplated are libraries of Imaging Strands, which libraries may contain, for instance: a set of ISs that have identical primary nucleotide sequences but that vary because each includes a different signal molecule (which may all be of a type, for instance, all fluorophores where FLIM is used as the measure of altered proximal environment; or of different types or detectable signals; and so forth); a set of ISs with variety as to primary nucleotide sequence (such as different length, different primary sequence, different sequences but equivalent proportion of purine/pyrimidine content, one or more modified nucleotides, varying overhang or underhang compared to the cognate DS, and so forth), but with the same signal molecule bound to each; and so forth. [0190] Also contemplated are unlabeled IS strands for use with a dye labeled minor groove binder (mgb). Mgb have sequence specificity (mostly A:T) design of DS attachment to NTAA and surrounding sequence context can position dye-mgb such that lifetime changes can be appreciated. In this embodiment, the dye is noncovalently attached to IS. Filed: January 4, 2024 [0191] The sequencing methods described herein are based around super resolution imaging (of changes in one or more spectral characteristic(s) of signal molecule(s), which characteristics are influenced based on proximity to different terminal amino acids of immobilized peptides), using DNA docking and Imaging strands. It may be advantageous to design ISs that can exchange or de-hybridize at a rate that is slower than fluorescence lifetime measurements in order to reduce or minimize signal decay from photobleaching. DNA-PAINT (Civitci et al., Nature Comm.11, Art. No. 4339, 2020, doi.org/10.1038/s41467-020-18181-6) can be employed with the provided peptide sequence methods. [0192] Methods of making both natural and modified (non-naturally occurring) oligonucleotides, which can be used to make imaging strand oligonucleoside for use in methods of the current disclosure, are well known in the art. Oligonucleotides are connected by an internucleotide bond that refers to a chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides may increase the binding affinity or stability oligonucleotides, or reduce the susceptibility of oligonucleotides nuclease digestion. Cationic modifications, including, but not limited to, diethyl-ethylenediamide (DEED) or dimethyl- aminopropylamine (DMAP) may be especially useful due to decrease electrostatic repulsion between the oligonucleotide and a target. Modifications of the phosphate backbone may also include the substitution of a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate internucleoside linkage in place of the phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo. [0193] Examples of modified nucleotides with reduced charge include modified internucleotide linkages such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g., Sterchak et al., Organic Chem., 52:4202, 1987), and uncharged morpholino-based polymers having achiral intersubunit linkages (see, for instance, U.S. Patent No. 5,034,506). Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles. [0194] In another embodiment, the oligonucleotides are composed of locked nucleic acids, or may contain at least one locked nucleic acid. Locked nucleic acids (LNA) are modified RNA nucleotides (see, for example, Braasch et al., Chem. Biol., 8(1):1-7, 2001). LNAs form hybrids with DNA which are more stable than DNA/DNA hybrids, a property similar to that of peptide nucleic acid (PNA)/DNA hybrids. Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some embodiments by adding positive charges to it. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs. Filed: January 4, 2024 [0195] In some embodiments, the oligonucleotides include peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the phosphate backbone of the oligonucleotide is replaced (for instance, in its entirety) by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are typically replaced by peptide bonds. The various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNAs maintain spacing of heterocyclic bases that is similar to conventional DNA oligonucleotides, but are achiral and neutrally charged molecules. Peptide nucleic acids are comprised of peptide nucleic acid monomers. [0196] Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone constituents of oligonucleotides such as PNA may be peptide linkages, or alternatively, they may be non-peptide peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like. Methods for the chemical assembly of PNAs are well known. See, for example, U.S. Pat. Nos.5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571. [0197] Oligonucleotides optionally include one or more terminal residues or modifications at either or both termini to increase stability, and/or affinity of the oligonucleotide for its target. Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. Oligonucleotides may further be modified to be end capped to prevent degradation using a propylamine group. Procedures for 3' or 5' capping oligonucleotides are well known in the art. (IX) Docking Strands, and Attachment of Docking Strand to Immobilized Peptides [0198] In embodiments, a docking strand (DS) is a synthetic single stranded DNA (ssDNA) with a chosen sequence and a modification (for instance, on the 3’ end of the oligonucleotide) that enables covalent linkage of the ssDNA to the free terminal amino acid of an immobilized protein, polypeptide, or peptide. In one example, the modification is an alkyl thiol group such as (3- mercaptopropyl)phosphate, which enables covalently linkage to the N-terminal maleimido group of a surface-conjugated MPITC-peptide. Though exemplifications are provided, embodiments are contemplated where other DNA oligo modifications are used - including at the 5’ end, internal within the oligo, or at the 3’ end. Internal modifications are generally limited to dT, abasic, or spacers. There are well recognized and commercially available types of oligo modification that can be employed; see, for instance, information provided online by Integrated DNA Technologies (see, for instance, resources available at idtdna.com/pages/products/custom-dna-rna/oligo- modifications). Filed: January 4, 2024 [0199] DS used in the provided methods can be “universal”, in that the oligonucleotide can be bound to any/all peptides in an experiment regardless of the primary sequence of the peptides. This universality is beneficial because it simplifies the system of analysis, since different docketing strands (or other binding moieties) are not required for associate with / labeling of immobilized peptides. The universal DS is not dependent on the primary sequence of the peptide(s) to be analyzed, and it can be bound to (covalently attached to) whatever terminal amino acid is present on each immobilized peptide. [0200] The DS is attached (chemically) to the TAA of a peptide/polypeptide (or one DS is attached to each TAA of an array of peptides/polypeptides) that is/are immobilized on a support surface. A single stranded region of the DS is available, to allow binding of an IS, in order to bring the signal molecule (e.g., fluorophore) attached to the IS into proximity with the TAA and its side chain. [0201] The DS sequence can be the same between each cleavage cycle or it can be changed to different/partially different sequence. The DS can be used for FLIM measurements to fingerprint amino acids. [0202] Examples are provided herein of linkers that are useful to attach a docketing strand to immobilized peptide(s) for use in protein/peptide analysis (sequence) methods. Generally, these linkers can be imagined as three-part structures, have a “DNA Attach” function on one end, a “Peptide Attach” function on the other, and some chemical bridge or other moiety that joins the two functional elements together. The “DNA Attach” portion is characterized as having a chemical structure capable of being joined (covalently) to a DNA molecule (particularly, a ssDNA DS), while the “Peptide Attach” portion is characterized as having a chemical structure capable of being joined (covalently) to the terminal end of a peptide molecule. Depending on the embodiment, the “Peptide Attach” portion of the linker is capable of being attached at the N-terminus of the peptide; from the C-terminus of the peptide. Similarly, depending on the embodiment, the “DNA Attach” portion of the linker is capable of being attached at the 5’ end of an oligo; at the 3’ end of an oligo; or at site(s) within the internal sequence of the oligo. Additional details and options are provided herein. [0203] Sample barcodes or UMIs can be incorporated in docking strands and decoded by sequential hybridization. Thus, the DS can also be used to encode other information, such as sample or spatial barcodes. For sample barcodes, the DS would be added to the peptides before immobilization on a surface. Different DS sequences attached to different samples. Then mixed together and immobilized on surface. Labeled imaging strands bind to complementary DS and report location and sample ID of individual peptides. If sample numbers are greater than dye colors, then DS can have multiple IS binding sequences and serial binding of isA and isB can demux. 3 colors, 2 IS sites = 3^2 = 9 sample barcodes, which is analogous to MER-fish. The Filed: January 4, 2024 barcode DS would ideally be cleaved and then FLIM DS attached for sequencing. Spatial barcoding is similar to sample barcodes but have higher number of combinations. 4^8 = 65K barcodes. Unlabeled IS can be a “color”. Real UMIs may be difficult to decode since there are so many molecules. [0204] Methods of making both natural and modified (non-naturally occurring) oligonucleotides, which can be used to make docking strand oligonucleoside for use in methods of the current disclosure, are well known in the art. Exemplary methods are provided herein. (X) Binding of Labeled Imaging Strand to Docking Strand on Immobilized Peptides [0205] Binding of the IS oligonucleotide (which is a ssDNA molecule) to the DS oligonucleotide (which is a ssDNA molecule) relies on conventional DNA double strand hydrogen bond formation. Thus, binding is carried out under art recognized conditions that enable pairing of the two ssDNA strands such that hydrogen bonds form between the bases adenine and thymine to form the AT base pair and between the bases guanine and cytosine to form the GC base pair. [0206] Those of ordinary skill in the art will understand that conditions can be varied to influence the annealing affinity, including modifying salt and other buffer conditions, temperature, and the like. The primary sequence of the two strands, including modifications and over- or under-hangs will also influence annealing. [0207] The melting temperature of the DS:IS complex will be influenced by the length of the IS and DS oligos, as well as the guanine and cytosine content and the environmental salt and other buffer conditions used in binding (and dissociation). DNA hybridization melting temperature (Tm) is affected by salt concentration in the reaction buffers and washes. If employing PAINT techniques then lower salt, higher temperature, and/or co-solvents can tune those kinetics. Similarly, dyes that have low aqueous solubility could benefit from having some solvent (DMSO, DMF, PEG) Washes to remove imaging strands can be improved through use of detergents (anionic, zwitterionic, cationic). [0208] It is also known that lifetime effects may be influenced by solvent conditions; see, e.g., Boens et al. (Anal Chem. 79(5):2137-2149, 2007, doi:10.1021/ac062160k). If employing DNA- PAINT techniques are being employed, then lower salt, higher temperature, and/or co-solvents can be used to tune those kinetics. Similarly, dyes that have low aqueous solubility could benefit from having some solvent (DMSO, DMF, PEG) Washes to remove imaging strands can be improved through use of detergents (anionic, zwitterionic, cationic). Filed: January 4, 2024 (XI) Detection of Imaging Strand Signal(s) [0209] Various statistical methods known in the art may be used to compare the spectra of a signal molecule in the proximity of different amino acids in order to identify the closest match and thereby identify the terminal amino acid (such as the N-terminal amino acid) of an immobilized polypeptide. [0210] In one embodiment, suitable methods generate a quantitative measure of similarity or difference between the measured spectra (e.g., fluorescence lifetime) and a reference spectrum (e.g., fluorescence lifetime), which for instance is the spectra obtained using the same signal molecule in the proximity of a known amino acid at the terminal position of an immobilized polypeptide. [0211] In embodiments, the methods further include generating a statistical measure or probability score that a spectrum is indicative of the presence of a particular terminal amino acid (e.g., a NTAA or CTAA, depending on the embodiment and method) adjacent to a signal molecule. In one embodiment, the methods used herein for comparing measured spectral property(s) of a signal molecule proximal to a test terminal amino acid and a reference/control terminal amino acid use one or more probabilistic algorithms. For example, a probabilistic algorithm can be trained to identify different N-terminal amino acids by associating specific spectra with specific, known N- terminal amino acids. As demonstrated herein, the N-1 and N-2 amino acids also may influence the spectra, and so this can be taken into account in developing reference/control measurements. [0212] Lifetime measurement is described herein in various embodiments with regard to measurement of the fluorescence lifetime of a fluorophore on the IS. However, other marker systems are envisioned, in which the signal “lifetime” of a different marker may be measured. For example, the imaging strand may be constructed so that luminescence or phosphorescence lifetime is measured. [0213] Systems that can perform single photon or multiphoton lifetime imaging (including time- domain and frequency-domain), including commercially available systems, may be able to perform these measurements and analyses employed in the methods described herein. Commercial systems include those from Becker & Hickl, PicoQuant, Olympus, Leica- microsystems, ISS, Nikon, and Zeiss. In-house systems require a pulsed laser source, fast electronics, and detector (photomultiplier tube, avalanche photodiode, metal oxide semiconductor sensor), as well as microscopy equipment in order to perform similar measurements discussed in this art. [0214] It is also noted that different signal molecules (including for instance fluorophores) may exhibit different spectral characteristics, depending on the buffer conditions and salt conditions under which spectral characteristics are measured. Thus, additional distinguishing information Filed: January 4, 2024 can be obtained by changing the conditions under which a spectral measurement is made. The fluorescence lifetime is extremely sensitive to the changes in immediate molecular environment. The factors affecting the fluorescence lifetime include pH, ion concentration, viscosity, hydrophobic properties, oxygen concentration. Oxygen can damage dyes so it would be mitigated by anti-photobleaching agents - commercially available solutions. [0215] Alternative spectroscopic techniques such as anisotropy or fluorescence polarization, may be used to enhance distinguishing amino acid residues along a peptide. [0216] DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super resolution technique that allows image resolution below the diffraction limit of light. In FLIM, the IS strand is stably bound to DS so all peptide locations have signal and must be spatially resolvable to be properly distinguished. This drives density or spacing of peptides on the support surface. In embodiments of peptide sequencing that employ PAINT, the IS is transiently bound to DS so each peptide location is “blinking” stochastically. Since a fraction of the peptides are ON at any given time, the spacing density can be increased below the normal resolution limit. Another benefit of PAINT is that the IS is being exchanged so photobleaching or dye problems are mitigated. A downside to employing PAINT is that only a fraction of the peptides is detected at any given time, so accumulation time increases to 5-15 minutes, for instance. The on/off or blinking rate of PAINT in such combined embodiments is slower than the fluorescence lifetime (or whatever other spectral characteristic(s) are being measured). [0217] Cleavage events can also be measured by re-interrogation, which provides error correction. Tracking errors during data collection, correcting during analysis. Accuracy is important in sequencing. Tracking NTAA cleavage will inform on phasing. IF DS/IS hybridization is negated after cleavage then monitor spot signal longitudinally. If a spot goes dark in a subsequent cycle, then either cleavage failed and DS is damaged or new DS conjugation failed. If it reappears in subsequent cycle, then either cleavage occurred or DS conjugation was successful. Won’t know which error happened but will know to run gap alignment models at that position. IF DS/IS hybridization is intact after cleavage then these approaches can be useful: A approach will be to hybridize IS after cleavage to determine which DS are still present – indicative of failed cleavage. These peptides are lagging. In practice could be difficult if cleavage is efficient, the number of positive spots will be low. This can present a problem with image registration. Fiducial markers can help address but may not solve the problem if there is image distortion. After new DS conjugation, spots that are still dark represent failed conjugation. Another approach is to use a different DS in the subsequent cycle and a different set of IS. Only the sequence changes, the dye panel can be same. Missing peptide signal relative to previous images would be due to failed cleavage or failed DS conjugation. To avoid doubling reagent #s, the DS can have a second Filed: January 4, 2024 binding region that is specifically for error checking. IS binding region remains constant. Alternate DS1 and DS2 between cycles, probe with IS1’ and IS2’ – preferably different colors – to track cleavage/conjugation outcomes at each cycle. Most spots (locations) will light up in this approach; those that remain dark indicate failed DS conjugation. Tracking peptide signals between cycles informs on error type, and will influence what alignment algorithms can be used to reconstruct sequence. [0218] It will be recognized by those of skill in the art, that buffer conditions such as pH, co- solvents, salts, metals, and the like can also influence TAA dependent spectral characteristic lifetime changes. Changing buffer/solvent conditions can be used to modulate interactions between the TAA side chain and the signal molecule on the IS. Additional data can therefore be gathered by interrogating the same peptide / DS / IS / signal molecule combination under different buffer conditions. For instance, salts and pH can affect ionic interactions between groups; solvents, surfactants, and so forth can affect other interactions (such as impacts on weak interactions, van der Wahls forces, hydrophobic, aromatic, ionic, and so forth). (XII) Cleavage of Terminal Amino Acid and Release of Docking Strand [0219] In certain embodiments relating to analyzing (e.g., sequencing) peptides, following binding of a terminal amino acid (N-terminal or C-terminal) by a binding agent and detecting the signal generated by binding of a labeled IS to a DS, the terminal amino acid is removed or cleaved from the peptide to expose a new terminal amino acid. Optionally, the labeled IS is dissociated from the DS and washed away before such cleavage. [0220] In some embodiments, the terminal amino acid is an NTAA. In other embodiments, the terminal amino acid is a CTAA. [0221] Cleavage of a terminal amino acid can be accomplished by any number of known techniques, including chemical cleavage and enzymatic cleavage or digestion. In some embodiments, an engineered enzyme that catalyzes or reagent that promotes the removal of the PITC-derivatized or other labeled N-terminal amino acid is used. In some embodiments, the terminal amino acid is removed or eliminated using any of the methods as described in US2020/0348307, WO2020/223133 or WO2020/198264. In some embodiments, cleavage of a terminal amino uses a carboxypeptidase, an aminopeptidase, a dipeptidyl peptidase, a dipeptidyl aminopeptidase or a variant, mutant, or modified protein thereof; a hydrolase or a variant, mutant, or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; anhydrous TFA, a base; or any combination thereof. In some embodiments, the mild Edman degradation uses a dichloro or monochloro acid; the mild Edman degradation uses TFA, TCA, or DCA; or the mild Edman degradation uses triethylamine, triethanolamine, or triethylammonium acetate Filed: January 4, 2024 (Et₃NHOAc). In some cases, the reagent for removing the amino acid includes a base. In some embodiments, the base is a hydroxide, an alkylated amine, a cyclic amine, a carbonate buffer, trisodium phosphate buffer, or a metal salt. Also contemplated are digestion using exo- or endo- peptidases. [0222] In some embodiments, the chemical reagent for removing a portion of the polypeptide is selected from a phenyl isothiocyanate (PITC), a nitro-PITC, a sulfo-PITC, a phenyl isocyanate (PIC), a nitro-PIC, a sulfo-PIC, Cbz-Cl (benzyl chloroformate) or Cbz-OSu (benzyloxycarbonyl N- succinimide), an anhydride, a 1-fluoro-2,4-dinitrobenzene (Sanger's reagent, DNFB), dansyl chloride (DNS-Cl, or 1-dimethylaminonaphthalene-5-sulfonyl chloride), 4-sulfonyl-2- nitrofluorobenzene (SNFB), 2-Pyridinecarboxaldehyde, 2-Formylphenylboronic acid, 2- Acetylphenylboronic acid, 1-Fluoro-2,4-dinitrobenzene, 4-Chloro-7-nitrobenzofurazan, Pentafluorophenylisothiocyanate, 4-(Trifluoromethoxy)-phenylisothiocyanate, 4- (Trifluoromethyl)-phenylisothiocyanate, 3-(Carboxylic acid)-phenylisothiocyanate, 3- (Trifluoromethyl)-phenylisothiocyanate, 1-Naphthylisothiocyanate, N-nitroimidazole-1- carboximidamide, N,N′-Bis(pivaloyl)-1H-pyrazole-1-carboxamidine, N,N′-Bis(benzyloxycarbonyl)- 1H-pyrazole-1-carboxamidine, an acetylating reagent, a guanidinylation reagent, a thioacylation reagent, a thioacetylation reagent, a thiobenzylation reagent, and a diheterocyclic methanimine reagent, or a derivative thereof. [0223] Enzymatic cleavage of a NTAA may be accomplished by a peptidase, e.g., a carboxypeptidase, aminopeptidase, or dipeptidyl peptidase, dipeptidyl aminopeptidase, or variant, mutant, or modified protein thereof. Aminopeptidases naturally occur as monomeric and multimeric enzymes, and may be metal or ATP-dependent. Aminopeptidases are enzymes that cleave amino acids from the N-terminus of proteins or peptides. Natural aminopeptidases have limited specificity, and generically cleave N-terminal amino acids in a processive manner, cleaving one amino acid off after another (Kishor et al., Anal. Biochem.488:6-8, 2015). However, residue specific aminopeptidases have been identified (Eriquez et al., J. Clin. Microbiol., 12:667-71, 1980; Wilce et al., Proc. Natl. Acad. Sci. USA 95:3472-3477, 1998; Liao et al., Prot. Sci.13:1802-10, 2004). [0224] For the methods described here, aminopeptidases (e.g., metalloenzymatic aminopeptidase) may be engineered to possess specific binding or catalytic activity to the NTAA only when modified with an N-terminal label. For example, an aminopeptidase may be engineered such than it only cleaves an N-terminal amino acid if it is modified by a group such as PTC, modified-PTC, Cbz, DNP, SNP, acetyl, guanidinyl, diheterocyclic methanimine, etc. In this way, the aminopeptidase cleaves only a single amino acid at a time from the N-terminus, and allows control of the degradation cycle. In some embodiments, the modified aminopeptidase is non- Filed: January 4, 2024 selective as to amino acid residue identity while being selective for the N-terminal label. In other embodiments, the modified aminopeptidase is selective for both amino acid residue identity and the N-terminal label. [0225] For embodiments relating to CTAA analysis, methods of cleaving CTAA from peptides are also known in the art. For example, U.S. Patent No.6,046,053 discloses a method of reacting the peptide or protein with an alkyl acid anhydride to convert the carboxy-terminal into oxazolone, liberating the C-terminal amino acid by reaction with acid and alcohol or with ester. Enzymatic cleavage of a CTAA may also be accomplished by a carboxypeptidase. [0226] In embodiments, the methods described herein include cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide using Edman, or related, chemical degradation. In one embodiment, the methods described herein include cleaving the N-terminal amino acid or N-terminal amino acid derivative enzymatically with a protease, for example an aminopeptidase. [0227] Edman degradation generally involves two steps, a coupling step and a cleaving step. These steps may be iteratively repeated, each time removing the exposed N-terminal amino acid residue of a polypeptide. In one embodiment Edman degradation proceeds by way of contacting the polypeptide with a suitable Edman reagent such as PITC, or an ITC-containing analogue, at an elevated pH to form a N-terminal thiocarbamyl derivative. Reducing the pH, such by the addition of trifluoroacetic acid results in the cleaving the N-terminal amino acid thiocarbamyl derivative from the polypeptide to form a free anilinothiozolinone (ATZ) derivative. Optionally, this ATZ derivative may be washed away from the sample. In one embodiment the pH of the sample is modulated in order to control the reactions governing the coupling and cleaving steps. [0228] In some embodiments, the N-terminal amino acid is contacted with a suitable Edman reagent such as PITC, or an ITC containing analogue, at an elevated pH prior to contacting the affixed polypeptide with a plurality of probes that selectively bind the N-terminal amino acid derivative. Optionally, the cleaving step includes reducing the pH in order to cleave the N-terminal amino acid derivative. [0229] In conventional Edman degradation, polypeptides are sequenced by degradation from their N-terminus using the Edman reagent, phenyl isothiocyanate (PITC). The process employs two steps: coupling and cleavage. In the first step (coupling), the N-terminal amino group of a peptide reacts with phenyl isothiocyanate to form a thiourea. In the second step, treatment of the thiourea with anhydrous acid (e.g., trifluoroacetic acid) results in cleavage of the peptide bond between the first and second amino acids. The N-terminal amino acid is released as a thiazolinone derivative. Filed: January 4, 2024 [0230] In embodiments, the removal of a terminal amino acid from peptides being analyzed may be carried out using an Edman degradation enzyme such as those discussed in U.S. Patent No. 10,852,305, which can be used for cleaving the N-terminal amino acid of a peptide or polypeptide). Such enzymes may catalyze the cleavage step of the Edman degradation in aqueous buffer and at neutral pH, thereby providing an alternative to the harsh chemical conditions typically employed in conventional Edman degradation. An example Edman degradation enzyme may be a modified cruzain enzyme (a “cruzipain”), where cruzain is a cysteine protease from the protozoa Trypanosoma cruzi. See, for instance, Santos et al. (Sci. Reports 11:18231, 2021). (XIII) Cyclic Methods and Applications [0231] Provided in the methods herein, following one cycle of contacting docking strands attached to the immobilized polypeptides with labeled ISs and signal detection, these steps may be repeated sequentially one or more times. Two types of cycle are envisioned: interrogation of the same terminal amino acid with a series of two or more different signal molecules on sequentially bound IS (which bind sequentially to the same DS), and signal detection for each; and removal of the terminal amino acid (with its attached DS), followed by attachment of DS to the newly exposed (next) terminal amino acid, binding of a labeled IS, and signal detection. Optionally, these two cycles can be combined – where each amino acid in an immobilized polypeptide is interrogated with a series of two or more different signal molecules (each attached to an IS), and then the terminal amino acid is removed and the next (newly exposed) terminal amino acid is interrogated with a series of two or more different signal molecules (each attached to an IS), and so forth. Each sequential terminal amino acid can be interrogated with the same set of different signal molecules, or with a different set; and the order of such interrogation may be the same or different. [0232] In some embodiments, the polypeptide method includes removing a portion of the polypeptide. In some embodiments, the method includes removing the terminal amino acid from the peptide (along with the attached DS), thereby yielding a newly exposed terminal amino acid. This newly exposed terminal amino acid can be attached to a new DS, which is then contacted with an IS labeled with a detection agent, and the signal (that is influenced by the local environment as that is influenced by the newly exposed terminal amino acid) detected – and thus sequence may be repeated on each newly exposed terminal amino acid. Removal of a portion of the polypeptide, e.g., a terminal amino acid such as a NTAA, may be accomplished by any number of known techniques, including chemical and enzymatic techniques (including those described and exemplified herein). In some embodiments, the repeated steps for analyzing the newly exposed NTAA are substantially similar to the first cycle, including attaching a docking strand to the newly exposed NTAA, binding to the newly attached DS at least one labeled IS, and Filed: January 4, 2024 detecting a signal (such as a spectral characteristics, for instance a lifetime measurement) generated by the signal molecule label in the proximal environment that is formed when the signal molecule is brought into proximity with newly exposed NTAA. In some cases, it may be beneficial to wash the polypeptide (for instance, by washing the solid surface on which the polypeptide has been immobilized) with, for example, a suitable buffer to remove and/or dissociate components between steps. [0233] Thus, in embodiments the NTAA of the polypeptide is cleaved (and the C-terminus of the polypeptide is immobilized on a support). Cleaving away the initial NTAA exposes the N-terminal amino group of an adjacent (penultimate) amino acid on the polypeptide, whereby the adjacent amino acid is the available for reaction with a DS – and thereby characterization of the identity of that amino acid. Optionally, the polypeptide is sequentially cleaved (each repetition of which may be considered a cycle) until the last amino acid in the polypeptide (C-terminal amino acid) is reached. However, fewer than all of the amino acids in the immobilized polypeptide may optionally be analyzed. [0234] In other embodiments, the CTAA of the polypeptide is cleaved (and the N-terminus of the polypeptide is immobilized on a support). Cleaving away the initial CTAA exposes the C-terminal carboxyl group of an adjacent (penultimate) amino acid on the polypeptide, whereby the adjacent amino acid is the available for reaction with a DS – and thereby characterization of the identity of that amino acid. Optionally, the polypeptide is sequentially cleaved (each repetition of which may be considered a cycle) until the last amino acid in the polypeptide (N-terminal amino acid) is reached. However, fewer than all of the amino acids in the immobilized polypeptide may optionally be analyzed. (XIV) Assembly of Polypeptide Sequence – Comparison to Database(s) [0235] While identification of amino acids as described herein employs new and inventive methods, compositions, and techniques, the process of assembling the resultant peptide sequences into proteins and their identification based on comparison to protein sequence databases and the like are generally conventional. For instance, methods for assembling peptide sequences based on other peptide analyses (such as mass spectrometery) are well known; see for instance Zhang et al. (Curr. Protoc. Mol. Biol. 108:10.12.1-10.21.30, 2014, doi.org/10.1002/0471142727.mb1021s108). [0236] A widely used approach does not attempt to directly extract peptide sequence information from the spectrum. Instead, it utilizes algorithms to match the experimental data against theoretical spectra, which are calculated for all peptides in the database. A score is assigned to each match to determine the confidence of the match. This approach is highly automated and Filed: January 4, 2024 best suited for high-throughput proteomic analysis of complex samples (Link et al., Nat Biotech. 17(7):676-682, 1999). A number of algorithms have been developed for this approach (Sadygov et al., Anal Chem.76(6):1664-1671, 2004; Kapp et al., Proteomics, 5(13):3475-3490, 2005). The most widely used algorithms are MASCOT (Perkins et al., Electrophoresis. 20(18):3551-3567, 1999), SEQUEST (Link et al., Nat Biotech.17(7):676-682, 1999), X!Tandem (Craig and Beavis, Bioinformatics.20(9):1466-1467, 2004), and OMSSA (Geer et al., J Proteome Res.3(5):958-965, 2004). [0237] In one embodiment of the description, the method includes comparing sequence information obtained for each polypeptide molecule to a reference protein sequence database. In some embodiments, small fragments of 10-40, or fewer, sequenced amino acid residues, consecutive or with gaps, may be useful for detecting the identity of a polypeptide in a sample. It has been demonstrated that protein sequencing can be accomplished by identifying only a subset of amino acids within a sequence, then comparing the partial or incomplete alignment with a database (e.g., sparse sequencing). See, for instance, Swaminathan et al., PloS Comput. Biol. 11(2):31004080, 2015, doi:10.1371/journal.pcbi.1004080; and Swaminathan et al., Nature Biotech.36:1075-1082, 2018). [0238] An application such as Proteome Discoverer (ThermoFisher) can align the peptide fragments from the above search algorithms to putative proteins. [0239] As will be understood by those having ordinary skill in the art, aspects of the present disclosure may be embodied as a system, method, or computer program product. As such, embodiments of the present disclosure may manifest as an entirely hardware embodiment, an entirely software embodiment (including firmware, resident software, micro-code, etc.), or an embodiment that combines both software and hardware aspects. These may all generally be referred to herein as a “circuit,” “engine,” “module,” or “system.” Furthermore, aspects of the present disclosure may take the form of a computer program product embodied in one or more computer readable medium(s) having computer readable program code instantiated upon them. [0240] Aspects of the present disclosure may be implemented using one or more analog and/or digital electrical or electronic components, and may include a microprocessor, a microcontroller, an application-specific integrated circuit, a field programmable gate array, programmable logic and/or other analog and/or digital circuit elements configured to perform various input/output, control, analysis, and other functions described herein, such as by executing instructions of a computer program product. [0241] The system for generating a database for diagnosing and treating a systemic inflammatory condition may include corresponding computer device, computer readable media, network, and remote device. Filed: January 4, 2024 [0242] The computing device may include, but is not limited to, a processor(s), memory, input and/or output devices, and a display device. The memory includes, but is not limited to one or more databases, an application for running a mass spectrometer and for analyzing mass spectrometer data, and a client facing application. The computing device may be accessed by a remote device via a network. [0243] In some instances, the information from the provided methods can be stored, analyzed, and/or determined using a software tool. The software may utilize information the binding characteristics of each binding agent. The software could also utilize a listing of some or all spatial locations in which each a signal was generated or not generated by the detectable label. In some embodiments, the software may include a database. The database may contain sequences of known proteins in the species from which the sample was obtained or also include related species (e.g., homologs). In some cases, if the species of the sample is unknown then a database of some or all protein sequences may be used. The database may also contain the characteristics and/or sequences of any known protein variants and mutant proteins thereof. [0244] In some embodiments, the software may include one or more algorithms, such as a machine learning, deep learning, statistical learning, supervised learning, unsupervised learning, clustering, expectation maximization, maximum likelihood estimation, Bayesian inference, linear regression, logistic regression, binary classification, multinomial classification, or other pattern recognition algorithm. For example, the software may perform the one or more algorithms to analyze the information regarding (i) spectral characteristic(s) of each signal molecule used, (ii) information from the database of proteins, and/or (iii) a list of locations observed (including in different cycles), in order to generate or assign a probable identity to each signal detected and/or a confidence (e.g., confidence level and/or confidence interval) for that information. (XV) Kits and Articles of Manufacture [0245] Also provided herein are kits and articles of manufacture that include components for polypeptide sequencing analysis using one of the methods described herein. In some embodiments, the kits further contain other reagents for treating and analyzing proteins, polypeptides, or peptides. The kits and articles of manufacture may include any one or more of the reagents and components used in the provided methods. In some embodiments, the kit includes one or more of support surface(s), docking strand oligonucleotide(s) (optionally, already functionalized for attachment to peptides to be analyzed), imaging strand oligonucleotide(s) (optionally modified by attachment of signal molecules), a processing or reaction compounds or solutions for use in a peptide sequence method. Filed: January 4, 2024 [0246] For instance, exemplary peptide sequencing method kits include a reagent pack that includes a combination of two or more of DS and IS libraries, conjugation buffers, hybridization buffers, wash buffers, and cleavage buffers. A flow cell kit may include flow cell / substrates for immobilization of the peptide(s) to be analyzed, terminal activation reagents (C-terminal or N- terminal, depending on the analysis type), and immobilization and wash/blocking buffers. Kits may optionally include components useful in polypeptide fragmentation, though commercially available peptide fragmentation kits and systems may also be employed. [0247] In some embodiments, the kit further includes reagents for preparing the proteins or polypeptides. Any combination of fractionation, enrichment, and subtraction methods, of the proteins may be performed. For example, the reagent(s) may be used to fragment or digest the proteins. In some cases, the kit includes reagents and components to fractionate, isolate, subtract, and/or enrich proteins (or peptides) to be analyzed. In some examples, the kits further includes a protease. In some embodiments, the kit includes a support surface on which one or more or polypeptides can be immobilized, and one or more reagents for immobilizing polypeptides (or peptides) on a support. [0248] In some embodiments, the kit also includes one or more buffers or reaction fluids useful for or necessary for any of the reactions to occur. Buffers such as wash buffers, reaction buffers, binding buffers, elution buffers and the like are known to those or ordinary skill in the arts. In some embodiments, the kits further include buffer(s), and one or more additional components to accompany other reagents described herein. The reagents, buffers, and other components may be provided in vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Any of the components of the kits may be sterilized and/or sealed. [0249] In addition to above-mentioned components, the subject kits may further include instructions for using the components of the kit to practice the subject methods, such as instructions for sample preparation, sequence obtention, and/or analysis of data obtained from the method(s). The kits described herein may also include other materials, such as those that may be deemed desirable from a commercial and user standpoint, including other buffers, diluents, filters, syringes, and/or package inserts with instructions for performing at least one of the methods described herein. [0250] Any of the above-mentioned kit components, and any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological reagents), agent, structure (e.g., support, surface, particle, or bead), reaction intermediate, reaction product, binding complex, or any other article of manufacture disclosed and/or used in the exemplary kits and methods, may be provided separately or in any suitable combination in order to form a kit. Filed: January 4, 2024 [0251] Also contemplated are devices useful to apply chemicals or compositions or washes/solutions to immobilization supports, and for exposing immobilized peptides to components of the provided methods (such as washes, buffers, reaction solutions, and the like), including flow throw fluid devices and micro-fluidic devices. [0252] Devices for detecting and measuring spectral characteristics of the analysed terminal amino acids, such as devices for detecting fluorescence lifetime, are also contemplated. [0253] Further embodiments are analysis software and amino acid deconvolution databases prepared using, or intended to be used with, any of the described peptide analysis/sequencing methods. (XVI) Representative Definitions [0254] To facilitate understanding, a number of terms are defined below. Terms used herein (unless otherwise specified) have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. The terminology herein is used to describe specific embodiments, but their usage is not intended to be limiting, except as outlined in the claims. [0255] The term “alkyl” refers to a straight or branched hydrocarbon. For example, an alkyl group can have 1 to 6 carbon atoms (i.e., C1-C6 alkyl or C1-6 alkyl), 1 to 4 carbon atoms (i.e., C1-C4 alkyl or C1-4 alkyl), or 1 to 3 carbon atoms (i.e., C1-C3 alkyl or C1-3 alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n- propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, - CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, - CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, - CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2- butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (- CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (- C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl (-CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (- CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (-C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl (- CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (-C(CH₃)₂CH(CH₃)₂), and 3,3-dimethyl-2-butyl (- CH(CH₃)C(CH₃)₃. [0256] The term “amino acid” in general refers to organic compounds that contain at least one amino group (-NH₂), and one carboxyl group (-COOH), where the carboxylic acids are deprotonated at neutral pH, having the basic formula of NH₂CHRCOOH. An amino acid and thus a peptide has an N (amino)-terminal residue region and a C (carboxy)-terminal residue region. Filed: January 4, 2024 The term “terminal” (referred to as singular terminus and plural termini) refers to the end position of a peptide or protein. The “N terminus” or N terminal amino acid is the one found at the amino den of the peptide, while the “C terminus” or C terminal amino acid is the one found at the carboxy end. The phrase “N-terminal amino acid” refers to an amino acid that has a free amine group and is only linked to one other amino acid by a peptide amide bond in the polypeptide. Optionally, the “N-terminal amino acid” may be an “N-terminal amino acid derivative”. As used herein, an “N- terminal amino acid derivative” refers to an N-terminal amino acid residue that has been chemically modified, for example by an Edman reagent or other chemical in vitro or inside a cell via a natural post-translational modification (e.g., phosphorylation) mechanism. [0257] Amino acids include the 20 standard, naturally occurring or canonical amino acids as well as non-standard amino acids. The standard, naturally-occurring amino acids include Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or Ile), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gln), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). An amino acid may be an L-amino acid or a D-amino acid. Non- standard amino acids may be modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or non-proteinogenic amino acids that occur naturally or are chemically synthesized. Examples of non-standard amino acids include, selenocysteine, pyrrolysine, and N-formylmethionine, β-amino acids, Homo-amino acids, Proline and Pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids. [0258] The terms “amino acid sequence”, “peptide”, “peptide sequence”, “polypeptide”, and “polypeptide sequence” are used interchangeably herein to refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide (amide) bond or an analog of a peptide bond. The term peptide includes oligomers and polymers of amino acids or amino acid analogs. The term peptide also includes molecules that are commonly referred to as peptides, which generally contain from two (2) to twenty (20) amino acids. The term peptide also includes molecules that are commonly referred to as polypeptides, which generally contain from twenty (20) to fifty amino acids (50). The term peptide also includes molecules that are commonly referred to as proteins, which generally contain from fifty (50) to three thousand (3000) amino acids. The amino acids of the peptide may be L-amino acids or D-amino acids. A peptide, polypeptide or protein may be synthetic, recombinant, or naturally occurring. A synthetic peptide is a peptide that is produced by artificial means in vitro. Filed: January 4, 2024 [0259] As used herein, “analyzing” a polypeptide means to identify, detect, quantify, characterize, distinguish, or a combination thereof, all or a portion of the components of the polypeptide. For example, analyzing a peptide, polypeptide, or protein includes determining all or a portion of the (contiguous or non-contiguous) amino acid sequence of the peptide. Analyzing a polypeptide also includes partial identification of a component of the polypeptide. For example, partial identification of amino acids in the polypeptide protein sequence can identify an amino acid in the protein as belonging to a subset of possible amino acids. Analysis typically begins with analysis of the n NTAA, and then proceeds to the next amino acid of the peptide (i.e., n−1, n−2, n−3, and so forth). This is accomplished by elimination of the n NTAA, thereby converting the n−1 amino acid of the peptide to an N-terminal amino acid (referred to herein as the “n−1 NTAA”). [0260] Analyzing a peptide may also include determining the presence, identification, and/or frequency of post-translational modifications on the peptide; and may optionally include information regarding the order of the post-translational modifications on the peptide, protein, or polypeptide. [0261] Analyzing a peptide may also include determining the presence and frequency of recognized characteristics of a protein, such as recognized structural and/or functional domains that are influenced by the primary (or secondary) sequence of the peptide, which may or may not include information regarding the sequential order or location of the domains within the polypeptide or peptide. Domains may include, for instance, epitopes in the peptide, which may or may not include information regarding the sequential order or location of the epitopes within the peptide. Analyzing the peptide may include combining different types of analysis, for example obtaining amino acid sequence information and post-translational modification information, or primary amino acid sequence and identification of domain(s). [0262] As used herein, the term “barcode” refers to a molecule providing a unique identifier tag or origin information for a polypeptide, a binding agent, a set of binding agents from a binding cycle, a sample polypeptides, a set of samples, polypeptides within a compartment (e.g., droplet, bead, or separated location), polypeptides within a set of compartments, a fraction of polypeptides, a set of polypeptide fractions, a spatial region or set of spatial regions, a library of polypeptides, or a library of binding agents. A “nucleic acid barcode” refers to a nucleic acid molecule of 2 to 30 bases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases). A “peptide barcode” or “amino acid barcode” refers to a sequence of amino acids that can have a length of at least, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 75, or 100 amino acids. A specific peptide barcode can be distinguished from other peptide barcodes by having a different length, sequence, or other physical property (for example, hydrophobicity). A barcode can be an Filed: January 4, 2024 artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the barcodes in a population of barcodes is different. A population of barcodes may be randomly generated or non- randomly generated. In certain embodiments, a population of barcodes are error-correcting or error-tolerant barcodes. Barcodes can be used to computationally deconvolute the multiplexed sequencing data and identify sequence reads derived from an individual polypeptide, sample, library, etc. [0263] The term “nucleic acid molecule” or “polynucleotide” refers to a single- or double- stranded polynucleotide containing deoxyribonucleotides or ribonucleotides that are linked by 3′- 5′ phosphodiester bonds, as well as polynucleotide analogs. A nucleic acid molecule includes DNA, RNA, and cDNA. A polynucleotide analog may possess a backbone other than a standard phosphodiester linkage found in natural polynucleotides and, optionally, a modified sugar moiety or moieties other than ribose or deoxyribose. Polynucleotide analogs contain bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence- specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide. Examples of polynucleotide analogs include xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), peptide nucleic acids (PNAs), γPNAs, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acid (TNA), 2′-O-Methyl polynucleotides, 2′-O-alkyl ribosyl substituted polynucleotides, phosphorothioate polynucleotides, and boronophosphate polynucleotides. A polynucleotide analog may possess purine or pyrimidine analogs, including for example, 7-deaza purine analogs, 8-halopurine analogs, 5- halopyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isocarbostyril analogues, azole carboxamides, and aromatic triazole analogues, or base analogs with additional functionality, such as a biotin moiety for affinity binding. In some embodiments, the nucleic acid molecule or oligonucleotide is a modified oligonucleotide. In some embodiments, the nucleic acid molecule or oligonucleotide is a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, a LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino DNA, or a combination thereof. In some embodiments, the nucleic acid molecule or oligonucleotide is backbone modified, sugar modified, or nucleobase modified. In some embodiments, the nucleic acid molecule or oligonucleotide has nucleobase protecting groups such as Alloc, electrophilic Filed: January 4, 2024 protecting groups such as thiranes, acetyl protecting groups, nitrobenzyl protecting groups, sulfonate protecting groups, or traditional base-labile protecting groups. [0264] The phrase “detectable label” refers to a substance which can indicate the presence of another substance when associated with it. The detectable label can be a substance that is linked to or incorporated into the substance to be detected. In some embodiments, a detectable label is suitable for allowing for detection and also quantification, for example, a detectable label that emitting a detectable and measurable signal. Detectable labels include any labels that can be utilized and are compatible with the provided polypeptide analysis assay format and include a bioluminescent label, a biotin/avidin label, a chemiluminescent label, a chromophore, a coenzyme, a dye, an electro-active group, an electro-chemiluminescent label, an enzymatic label, a fluorescent label, a latex particle, a magnetic particle, a metal, a metal chelate, a phosphorescent dye, a protein label, a radioactive element or moiety, and a stable radical. In provided embodiments, fluorescent labels are preferred. [0265] Direct and indirect attachments (e.g., of a detectable label to an oligonucleotide or other substance) can include covalent bonds or non-covalent interactions. Covalent bonds include the sharing of electrons in a chemical bond. Non-covalent interactions include dispersed electromagnetic interactions such as hydrogen bonds (such as occurs between paired strands of nucleic acids), ionic bonds, van der Waals interactions, and hydrophobic bonds. [0266] “Fluorescence” refers to the emission of visible light by a substance that has absorbed light of a different wavelength. In some embodiments, fluorescence provides a non-destructive means of tracking and/or analyzing biological molecules based on the fluorescent emission at a specific wavelength. Proteins (including antibodies), peptides, nucleic acid, oligonucleotides (including single stranded and double stranded primers), and so forth may be “labeled” with any of a variety of extrinsic fluorescent molecules referred to as fluorophores. Isothiocyanate derivatives of fluorescein, such as carboxyfluorescein, are an example of fluorophores that may be conjugated to proteins (such as antibodies for immunohistochemistry) or nucleic acids. In some embodiments, fluorescein may be conjugated to nucleoside triphosphates and incorporated into nucleic acid probes (such as “fluorescent-conjugated primers”) for in situ hybridization. [0267] The terms “individual” or “subject” include birds (e.g., chickens, ducks, geese, turkeys, quail, songbirds, and so forth), other non-mammalian vertebrates (e.g., as fish), and mammals (e.g., mice, rats, rabbits, and other rodents; cats and other felines; dogs and other canines; other domesticated animals; pigs, cows, oxen, sheep, goats, horses, and other livestock animals; monkeys and other non-human primates). In embodiments, the individual or subject is a human. [0268] The term “linker” refers to one or more of a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide, a polymer, or a non-nucleotide chemical moiety that is used to join Filed: January 4, 2024 two molecules to each other. A linker may be used to join a nucleic acid (such as a DS) with a polypeptide, a polypeptide with a support, a detection agent with a nucleic acid (such as an IS), and so forth. In certain embodiments, a linker joins two molecules via enzymatic reaction or chemistry reaction (e.g., click chemistry). [0269] As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times)—this depth of coverage is referred to as “deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, Thermo-Fisher, and Roche, including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays (see e.g., Service, Science 311:1544-1546, 2006). [0270] As used herein, “single molecule sequencing” or “third generation sequencing” refers to next-generation sequencing methods wherein reads from single molecule sequencing instruments are generated by sequencing of a single molecule, generally a molecule of DNA. Unlike next generation sequencing methods that rely on amplification to clone many DNA molecules in parallel for sequencing in a phased approach, single molecule sequencing interrogates single molecules (e.g., of DNA) and does not require amplification or synchronization. Single molecule sequencing includes methods that need to pause the sequencing reaction after each base incorporation (‘wash-and-scan’ cycle) and methods which do not need to halt between read steps. Examples of single molecule sequencing methods include single molecule real-time sequencing (Pacific Biosciences), nanopore-based sequencing (Oxford Nanopore), duplex interrupted nanopore sequencing, and direct imaging of DNA using advanced microscopy. [0271] The term “sample” refers to anything which may contain an analyte (e.g., a protein or peptide) for which an analyte assay (e.g., detecting, quantifying, and/or sequencing) is desired. The term “sample” can include a solution, a suspension, liquid, powder, a paste, any of which may be aqueous or non-aqueous, or any combination thereof. The sample may be a biological sample, such as a biological fluid or a biological tissue, or individual cell(s). Examples of biological Filed: January 4, 2024 fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid and the like. Biological tissues are aggregate of cells, usually of a particular kind (or a mixture of two or more kinds) together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective tissue, epithelium, muscle tissue, and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, and arteries. In some embodiments, the sample can be derived from a tissue or a body fluid, for example, a connective, epithelium, muscle or nerve tissue; a tissue selected from the group consisting of brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels; or a body fluid selected from the group consisting of blood, urine, saliva, bone marrow, sperm, an ascitic fluid, and subfractions thereof, e.g., serum or plasma. [0272] As used herein, the term “post-translational modification” refers to modifications that occur on a peptide after its translation, e.g., translation by ribosomes, is complete. A post- translational modification may be a covalent chemical modification or enzymatic modification. Examples of post-translation modifications include acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, C-terminal amidation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, farnesylation, flavin attachment, formylation, gamma-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S- sulfenylation, selenation, succinylation, sulfination, and ubiquitination. A post-translational modification includes modifications of the amino terminus and/or the carboxyl terminus of a peptide. Modifications of the terminal amino group include des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A post-translational modification also includes modifications, such as those described above, of amino acids falling between the amino and carboxy termini. The term post-translational modification can also include peptide modifications that include one or more detectable labels. [0273] The term “proteome” includes the entire set of proteins, polypeptides, or peptides (including conjugates or complexes thereof) expressed by a genome, cell, tissue, or organism at a certain time, of any organism. In one aspect, it is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. For example, a “cellular proteome” Filed: January 4, 2024 may include the collection of proteins found in a particular cell type under a particular set of environmental conditions, such as exposure to hormone stimulation. An organism's complete proteome may include the complete set of proteins from all of the various cellular proteomes. A proteome may also include the collection of proteins in certain sub-cellular biological systems. For example, all of the proteins in a virus can be called a viral proteome. As used herein, the term “proteome” include subsets of a proteome, including a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post- translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and/or nitrosylation), such as a phosphoproteome (e.g., phosphotyrosine-proteome, tyrosine-kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with a tissue or organ, a developmental stage, or a physiological or pathological condition; a proteome subset associated a cellular process, such as cell cycle, differentiation (or de-differentiation), cell death, senescence, cell migration, transformation, or metastasis; or any combination thereof. [0274] Proteomics is the study of a proteome. Thus, the term “proteomics” encompasses quantitative analysis of the proteome within cells, tissues, and bodily fluids, and the corresponding spatial distribution of the proteome within the cell and within tissues. Additionally, proteomics studies include the dynamic state of the proteome, which is continually changing in time as a function of biology and defined biological or chemical stimuli. [0275] In embodiments provided herein, the term “sample” includes any material that contains one or more polypeptides. The sample may be a biological sample, such as animal or plant tissue, biopsy, organ, cell(s), membrane vesicles, plasma membranes, organelles, cell extracts, secretions, urine or mucous or other secretion, tissue extracts or other biological specimens both natural or synthetic in origin. The term sample also includes single cells, organelles or intracellular materials isolated from a biological specimen, or viruses, prions, bacteria, fungus or isolates therefrom. The sample may also be an environmental sample, such as a water sample or soil sample, or a sample of any artificial or natural material, that contains one or more polypeptides. [0276] The term “side chains” or “R” (or R group) refers to unique structures attached to the alpha carbon (attaching the amine and carboxylic acid groups of the amino acid) that render uniqueness to each type of amino acid. R groups have a variety of shapes, sizes, charges, and reactivities, such as Charged Polar side chains, either positively or negatively charged, such as lysine (+), arginine (+), Histidine (+), aspartate (-) and glutamate (-), amino acids can also be basic, such as lysine, or acidic, such as glutamic acid; Uncharged Polar side chains have Hydroxyl, Amide, or Thiol Groups, such as Cysteine having a chemically reactive side chain, i.e. a thiol group that can form bonds with another Cysteine, Serine (Ser) and Threonine (Thr), that Filed: January 4, 2024 have hydroxylic R side chains of different sizes; Asparagine (Asn), Glutamine (Gln), and Tyrosine (Tyr); Non-polar hydrophobic amino acid side chains include the amino acid Glycine; Alanine, Valine, Leucine, and Isoleucine having aliphatic hydrocarbon side chains ranging in size from a methyl group for alanine to isomeric butyl groups for Leucine and Isoleucine. Methionine (Met) has a thiol ether side chain, Proline (Pro) has a cyclic pyrrolidine side group. Phenylalanine (with its phenyl moiety) (Phe) and Tryptophan (Trp) (with its indole group) contain aromatic side groups, which are characterized by bulk as well as non-polarity. [0277] As used herein, the terms “solid support”, “solid surface”, or “solid substrate”, or “sequencing substrate” refers to any solid material, including porous and non-porous materials, to which a polypeptide can be associated directly or indirectly, by any means known in the art, including covalent and non-covalent interactions, or any combination thereof. A solid support may be two-dimensional (e.g., planar surface) or three-dimensional (e.g., gel matrix or bead). A solid support can be any support surface including a bead, a microbead, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, a PTFE membrane, a PTFE membrane, a nitrocellulose membrane, a nitrocellulose-based polymer surface, nylon, a silicon wafer chip, a flow through chip, a flow cell, a biochip including signal transducing electronics, a channel, a microtiter well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a polymer matrix, a nanoparticle, or a microsphere. Materials for a solid support include acrylamide, agarose, cellulose, dextran, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polyester, polymethacrylate, polyacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, poly vinyl alcohol (PVA), Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polyvinylchloride, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, dextran, or any combination thereof. Solid supports further include thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers such as tubes, particles, beads, microspheres, microparticles, or any combination thereof. [0278] For example, when solid surface is a bead, the bead can include a ceramic bead, a polystyrene bead, a polymer bead, a polyacrylate bead, a methylstyrene bead, an agarose bead, a cellulose bead, a dextran bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a controlled pore bead, a silica-based bead, or any combinations thereof. A bead may be spherical or an irregularly shaped. A bead or support may be porous. A bead's size may range from nanometers, e.g., 100 nm, to millimeters, e.g., 1 mm. In certain embodiments, beads range in size from 0.2 micron to 200 microns, or from 0.5 micron to 5 micron. In some embodiments, beads can be 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, Filed: January 4, 2024 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, or 20 μm in diameter. In certain embodiments, “a bead” solid support may refer to an individual bead or a plurality of beads. In some embodiments, the solid surface is a nanoparticle. In certain embodiments, the nanoparticles range in size from 1 nm to 500 nm in diameter, for example, between 1 nm and 20 nm, between 1 nm and 50 nm, between 1 nm and 100 nm, between 10 nm and 50 nm, between 10 nm and 100 nm, between 10 nm and 200 nm, between 50 nm and 100 nm, between 50 nm and 150, between 50 nm and 200 nm, between 100 nm and 200 nm, or between 200 nm and 500 nm in diameter. In some embodiments, the nanoparticles can be 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 300 nm, or 500 nm in diameter. In some embodiments, the nanoparticles are less than 200 nm in diameter. [0279] The most widely used reaction for the sequential analysis of N-terminal residue of peptides is the Edman degradation method (Edman et al., Acta Chem. Scand.4: 283-293, 1950). Edman degradation is a method of sequencing amino acids in a peptide (or protein) wherein the amino- terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues). In the Edman procedure, phenyl isothiocyanate (PITC) reacts quantitatively with the free amino group of a peptide to yield the corresponding phenylthiocarbamoyl peptide. On treatment with anhydrous acid, the N-terminal residue is split off as a phenylthiocarbamoyl amino acid; this leaves the remainder of the peptide chain intact. One aspect of the Edman degradation method is that the rest of the peptide chain (after removal of the N-terminal amino acid) is left intact for further cycles of this procedure; thus the Edman method can be used in a sequential, iterative manner to identify a plurality of consecutive amino acid residues starting from the N-terminal end the peptide being analyzed. [0280] “Universal” docking strand as the phrase is used herein refers to a single-stranded DNA oligonucleotide that can be bound to any peptide, regardless of the primary sequence of the peptide. [0281] The Exemplary Embodiments and Examples below are included to demonstrate embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure. (XVII) Exemplary Embodiments First Embodiment Set [0282] 1. A method of sequencing a peptide have an initial N-terminal amino acid (NTAA), including sequential interrogation of the initial NTAA using a library of at least two different combinations of a ssDNA docking strand (DS) and a ssDNA imaging strand (IS), where a Filed: January 4, 2024 fluorophore is conjugated to the IS, to produce a set of fluorescence lifetime data having a characteristic fingerprint for each different amino acid of the peptide; wherein each pair of IS and DS are at least partially complementary in sequence. [0283] 2. The method of embodiment 1, wherein the sequential interrogation includes detecting and/or measuring interaction between the fluorophore and nucleobase(s) at or near the NTAA by detecting fluorescence lifetime data for each pair IS and DS in the library, for instance using fluorescence lifetime imaging (FLIM) single-photon fluorescence measurements. [0284] 3. The method of embodiment 1 or embodiment 2, further including removing the initial NTAA the peptide by an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. [0285] 4. The method of any one of embodiments 1-3, wherein the method is repeated for each subsequent amino acid in the peptide to produce a matrix of fluorescence lifetime data. [0286] 5. The method of embodiment 4, wherein the data is input into a machine learning algorithm to reconstruct a polypeptide sequence. [0287] 6. The method of any one of embodiments 1-6, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary so that spatial positioning and/or degrees of freedom of the attached fluorophore, in order to modulate interactions with the NTAA side chain, and thereby modulate the measured fluorescence lifetime. [0288] 7. The method of embodiment 6, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary by one or more of: inclusion of a modified nucleotide, inclusion of a non-natural nucleotide, inclusion of a 5’ IS overhang relative to the cognate DS, or inclusion of a 5’ IS underhang relative to the cognate DS. [0289] 8. The method of any one of embodiment 1-7, wherein the fluorophore includes Alexa Fluor® 88 (AF488), BODIPY-FL, BODIPY-TR, or TAMRA. [0290] 9. The method of any one of embodiments 1-8, wherein the fluorophore is conjugated at an end of the IS. [0291] 10. The method of any one of embodiments 1-8, wherein the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide within the IS (FIG.11). [0292] 11. The method of any one of embodiments 1-10, wherein removal of a N NTAA is performed under conditions such that the remaining peptide has a new N-terminal amino acid. [0293] 12. The method of any one of embodiment 1-11, wherein the peptide is immobilized on a solid support. [0294] 13. A database containing the matrix of fluorescence lifetime data of embodiment 4. Filed: January 4, 2024 [0295] 14. A method for identifying a N-terminal amino acid (NTAA) of a peptide, the method including: binding the C-terminal amino acid of the peptide to a solid surface; attaching to the NTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. [0296] 15. The method of embodiment 14, further including: cleaving the initial NTAA from the peptide, to leave a next NTAA of the peptide. [0297] 16. The method of embodiment 15, wherein cleaving the initial NTAA includes an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. [0298] 17. The method of any one of embodiments 14-16, including repeating the method a plurality of time to identify a sequence of the peptide. [0299] 18. A method of sequencing peptides, including: attaching peptide(s) to be sequenced to a solid substrate by their C-terminus; functionalizing the initial N-terminal amino acid of the immobilized peptides with a universal docking strand (DS) ssDNA oligo; contacting the DSs with an imaging strand (IS) oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule fluorescence lifetime (FLIM) measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; and cleaving the initial N-terminal amino acid from the peptide to reveal a second N-terminal amino acid; and optionally, carrying out another cycle of analysis for the second N-terminal amino acid. [0300] 19. A method of sequence a peptide, essentially as described herein. [0301] 20. A kit for carrying out the method of any one of embodiments 1-18, including at least one pair of IS and DS. [0302] 21. The kit of embodiment 20, including at least two pairs of IS and DS, where the two pairs differ by the fluorophore contained in the IS or by sequence, or both. Second Embodiment Set [0303] 1. A method for identifying a terminal amino acid (TAA) of a peptide having a N-terminal amino acid (NTAA) and a C-terminal amino acid (CTAA), the method including: binding either the NTAA of the peptide or the CTAA of the peptide to a solid surface to produce a bound TAA; attaching to the non-bound TAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting Filed: January 4, 2024 fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial TAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. [0304] 2. A method of sequencing a peptide having an initial terminal amino acid (TAA), including: interrogation of the initial TAA using a single-stranded DNA (ssDNA) docking strand (DS) attached to the initial TAA and a ssDNA imaging strand (IS) to which a signal molecule is conjugated, to produce a measurement of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide; wherein the IS and DS are at least partially complementary in sequence. [0305] 3. The method of embodiment 2, further including: sequential interrogation of the initial TAA using a library of at least two different combinations of a single-stranded DNA (ssDNA) docking strand (DS) and a ssDNA imaging strand (IS), where a signal molecule is conjugated to the IS, to produce a set of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide. [0306] 4. The method of embodiment 2, wherein the initial TAA is: the amino-terminal (N-terminal) amino acid (NTAA) of the peptide; or the carboxy-terminal (C-terminal) amino acid (CTAA) of the peptide. [0307] 5. The method of embodiment 2, wherein the method is carried out in parallel on a plurality of peptides. [0308] 6. The method of embodiment 2, wherein the signal molecule includes a fluorophore, and the spectral characteristic includes a measure of fluorescence. [0309] 7. The method of embodiment 6, wherein the spectral characteristic includes fluorescence lifetime. [0310] 8. A method of sequencing a peptide having an initial N-terminal amino acid (NTAA) and a C-terminal amino acid (CTAA), the method including: sequential interrogation of the initial NTAA using a library of at least two different combinations of a ssDNA docking strand (DS) and a ssDNA imaging strand (IS), where a fluorophore is conjugated to the IS, to produce a set of fluorescence lifetime data having a characteristic measurement for each combination of DS, IS, and fluorophore, wherein each pair of IS and DS is at least partially complementary in sequence. [0311] 9. The method of any one of embodiments 1-8, wherein the DS is a universal DS. [0312] 10. The method of any one of embodiments 1-8, wherein the interrogation or the sequential interrogation includes detecting and/or measuring interaction between fluorophore and amino acid sidechain at or near the CTAA or the NTAA by detecting fluorescence lifetime data for each of a plurality of IS / DS pairs in the library. Filed: January 4, 2024 [0313] 11. The method of embodiment 10, wherein detecting or measuring the interaction includes obtaining fluorescence lifetime imaging (FLIM) single-molecule fluorescence measurements for each of a plurality of IS / DS pairs in the library. [0314] 12. The method of embodiment 1 or embodiment 4, further including removing the initial CTAA or NTAA of the peptide by an Edman degradation reaction, enzymatic digestion, or a similar process. [0315] 13. The method of any one of embodiments 1-8, wherein the method is repeated for at least two subsequent amino acids in the peptide to produce a matrix of fluorescence lifetime data. [0316] 14. The method of embodiment 13, wherein the method is completed for each subsequent amino acid in the peptide to produce a matrix of fluorescence lifetime data. [0317] 15. The method of embodiment 13, wherein the fluorescence lifetime data is input into a machine learning algorithm to reconstruct a polypeptide sequence. [0318] 16. The method of embodiment 14, wherein the fluorescence lifetime data is input into a machine learning algorithm to reconstruct a polypeptide sequence. [0319] 17. The method of embodiment 3 or embodiment 8, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary so that spatial positioning and/or degrees of freedom of the attached fluorophore varies, in order to modulate interactions with the CTAA or NTAA side chain, and thereby modulate the measured fluorescence lifetime. [0320] 18. The method of embodiment 17, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary by one or more of: inclusion of a modified nucleotide, inclusion of a non-natural nucleotide, inclusion of a 5’ IS overhang relative to the cognate DS, or inclusion of a 5’ IS underhang relative to the cognate DS. [0321] 19. The method of embodiment 10, wherein the interaction between the CTAA or NTAA is further influenced by one or more of DS position, degrees of freedom, or another variable described herein. [0322] 20. The method of any one of embodiment 1, embodiment 6, or embodiment 8, wherein the fluorophore includes Alexa Fluor® 488 (AF488), BODIPY-FL, BODIPY-TR, TAMRA, or a KU dye. [0323] 21. The method of embodiment 20, wherein the fluorophore is conjugated at an end of the IS. [0324] 22. The method of any one of embodiments 1-8, wherein: the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide within the IS; or the peptide is conjugated to a modified nucleotide at or near an end of the IS and the fluorophore is conjugated to a modified nucleotide within the IS; or the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide Filed: January 4, 2024 at or near an end of the IS; or the peptide is conjugated to a modified nucleotide at or near an end of the DS and the fluorophore is conjugated to a modified nucleotide at or near an end of the IS. [0325] 23. The method of embodiment 12, wherein removing a CTAA or a NTAA is performed under conditions such that the remaining peptide has a new terminal amino acid available for another cycle of analysis. [0326] 24. The method of any one of embodiment 1-8, wherein the peptide or each peptide is immobilized on a solid support. [0327] 25. A database containing the matrix of fluorescence lifetime data of embodiment 14. [0328] 26. A method for identifying a N-terminal amino acid (NTAA) of a peptide having a NTAA and a C-terminal amino acid (CTAA), the method including: binding the CTAA of the peptide to a solid surface; attaching to the NTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. [0329] 27. A method for identifying a C-terminal amino acid (CTAA) of a peptide having a CTAA and a N-terminal amino acid (NTAA), the method including: binding the NTAA of the peptide to a solid surface; attaching to the CTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. [0330] 28. The method of embodiment 26 or embodiment 27, further including: cleaving the initial TAA from the peptide, to leave a next TAA of the peptide. [0331] 29. The method of embodiment 28, wherein cleaving the initial NTAA includes an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. [0332] 30. The method of embodiment 27 or embodiment 27, including repeating the method a plurality of times to identify a sequence of the peptide. [0333] 31. A method of sequencing peptides, each of which has a C-terminal amino acid (CTAA) and a N-terminal amino acid (NTAA), the method including: attaching peptide(s) to be sequenced to a solid substrate by their C-terminus, to form immobilized peptides; functionalizing the initial N- terminal amino acid of the immobilized peptides with a universal docking strand (DS) ssDNA oligo; contacting the DSs with an imaging strand (IS) oligo complementary to the DS oligo, which IS is Filed: January 4, 2024 conjugated to a first fluorophore; obtaining a single molecule fluorescence lifetime (FLIM) measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; cleaving the initial N-terminal amino acid from the peptide to reveal a second N-terminal amino acid; and optionally, carrying out another cycle of analysis for the second N-terminal amino acid. [0334] 32. A method of sequencing peptides, each of which has a C-terminal amino acid (CTAA) and a N-terminal amino acid (NTAA), the method including: attaching peptide(s) to be sequenced to a solid substrate by their N-terminus, to form immobilized peptides; functionalizing the initial C- terminal amino acid of the immobilized peptides with a universal docking strand (DS) ssDNA oligo; contacting the DSs with an imaging strand (IS) oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule fluorescence lifetime (FLIM) measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; cleaving the initial C-terminal amino acid from the peptide to reveal a second C-terminal amino acid; and optionally, carrying out another cycle of analysis for the second C-terminal amino acid. [0335] 33. A method of sequencing a peptide, essentially as described herein. [0336] 34. The method of embodiment 33, wherein the method includes detecting at least one spectral characteristic of a signal molecule, where the spectral characteristic is not fluorescence lifetime. [0337] 35. A kit for carrying out the method of any one of embodiments 1-34, including at least one pair of IS and DS. [0338] 36. The kit of embodiment 35, including at least two pairs of IS and DS, where the two pairs differ by the fluorophore contained in the IS or by sequence, or both.

Filed: January 4, 2024 [0339] 37. A compound of Formula (II) or a salt, or solvate is selected from the group consisting of C1- - - - - -C=OOR, -SO3, or any other common electron withdrawing groups; R¹ and R² are independently selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, -O-(C1-C6 alkyl), C1-C6 alkyl, hydroxy, halogen, -O- alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, - N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups. [0340] 38. The compound of embodiment 37 or a salt or solvate thereof, wherein x is 0. [0341] 39. The compound of embodiment 37 or a salt or solvate thereof, wherein y is 0. [0342] 40. The compound of embodiment 38 or a salt or solvate thereof, wherein y is 0. [0343] 41. The compound of any of embodiments 37-40, wherein: the C₁-C₆ alkyl of R¹ or R² is methyl, and the -O-(C₁-C₆ alkyl) of R¹ or R² is methoxy.

Filed: January 4, 2024 [0344] 42. The compound of embodiment 37, having the structure: ; wherein R₁ and R₂ are and OCH₃; with the proviso that R₁ and R₂ are the same; or a or [0345] 43. The compound of embodiment 42, which is (4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. [0346] 44. A process for preparing a compound of Formula (I) Filed: January 4, 2024 [0347] or a salt or solvate thereof, including converting a compound of Formula (III) [0348] to a compound a , and thereafter or solvate thereof to the compound of Formula (I) or a salt or solvate thereof, wherein: x is 0, 1 or 2; each R independently is selected from the group consisting of C1-C6 alkyl, -NO2, halogen, -C=OR, -C=SR, -C=ONR, - C=OOR, -SO3, or any other common electron withdrawing group; R¹ and R² are independently selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, -O-(C1-C6 alkyl), halogen, - O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating group; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, halogens, -O-alkyl, -S-alkyl, -O-C(=O)R, - Filed: January 4, 2024 N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating group. [0349] 45. The process of embodiment 44, wherein the compound of Formula (III) or the salt or solvate thereof is first converted to a compound of Formula (IV) or solvate thereof, followed by conversion of the the salt or solvate thereof to the compound of Formula (II) or solvate thereof. [0350] 46. The process of embodiment 45, wherein the conversion of the compound of Formula (III) of the salt or solvate thereof to the compound of Formula (IV) or the salt or solvate thereof takes place by reacting carbon disulfide (CS2) with the compound of Formula (III). [0351] 47. The process of embodiment 46, wherein the reaction takes place in the presence of a base. [0352] 48. The process of embodiment 47, wherein the base is a (C1-C6 alkyl)3N. [0353] 49. The process of embodiment 48, wherein the (C1-C6 alkyl)3N is triethylamine. [0354] 50. The process of embodiment 45, wherein the conversion of compound of Formula (IV) or the salt or solvate thereof to the compound of Formula (II) or the salt or solvate thereof takes place by reacting the compound of Formula (IV) or the salt or solvate thereof with di-tert-butyl carbonate (O-(C(=O)-OC(CH3)2)2). [0355] 51. The process of embodiment 50, wherein the reaction takes place in the presence of one or more bases. [0356] 52. The process of embodiment 51, wherein the one or more bases include dimethyl aminopyridine (DMAP) and triethylamine. Filed: January 4, 2024 [0357] 53. The process of embodiment 45, wherein the compound has the structure: ; wherein R₁ and R₂ are and OCH₃; with the proviso that R₁ and R₂ are the same; or a or [0358] 54. The process of embodiment 53, wherein the compound is (4-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. [0359] 55. Use of the compound of any one of embodiments 37-54 in a peptide analysis as described herein. (XVIII) Experimental Examples Example 1: Peptide Sequencing through Detection of Fluorescence Lifetime Perturbations [0360] Proof of concept of the workflow of fluorescence lifetime-based peptide sequencing (FIGs. 1A-1G) has been performed and has shown that the fluorescence lifetime can be affected by the chosen sequence of the DNA imaging strand (IS), including synthetic modifications to the nucleobase or sugar-phosphate backbone; the chosen fluorophore; and the identity of the N- terminal amino acid (NTAA), including intrinsic chemical variations or post-translational modifications (PTMs) of the NTAA side chain. This approach is agnostic to the chemical identity of the NTAA, so in addition to proteinogenic amino acids and their associated PTMs, it can also detect and identify non-canonical or unnatural amino acids. The NTAA was repeatedly interrogated by different IS-fluorophore conjugates, which can vary in either or both IS sequence and fluorophore to yield a set of fluorescence lifetime data. Edman degradation was performed to show that the NTAA can be removed to yield a new NTAA, which can then be interrogated to yield fluorescence lifetime data. This new NTAA can be repeatedly interrogated by different IS- fluorophore conjugates to yield a set of fluorescence lifetime data. This workflow can be Filed: January 4, 2024 performed repeatedly until all or a subset of the amino acids within the polypeptide chain have been interrogated. Edman degradation can be performed sequentially to sequentially expose amino acids of the polypeptide chain at the N-terminal position. All or a subset of these new NTAAs can be interrogated with the IS-fluorophore conjugates to generate data corresponding to the identity of the NTAA. Tthe positioning of each amino acid within the peptide chain is known based on the number of Edman degradation cycles performed. [0361] In the examples presented, synthetic peptides were used as a proxy for naturally occurring peptides or the component peptides of enzymatically or chemically digested proteins. These synthetic peptides were attached to a glass surface via the sulfhydryl group of a C-terminal cysteine, but the C-terminus can alternatively be covalently attached to the surface via the C- terminal carboxyl group. This can achieved by preparing a amine-functionalized surface, such as by silanization with (3-aminopropyl)triethoxysilane (APTES, CAS registry number: 919-30-2, IUPAC: 3-(triethoxysilyl)propan-1-amine), and covalently attaching the C-terminal carboxyl of a peptide to it using a coupling agent such as a carbodiimide (e.g. N,N’-diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) or N-hydroxysuccinimide (NHS). To perform this strategy consistently and with minimal side reactions, then amino, hydroxyl, and carboxyl-containing side chains could first be protected. Furthermore, the N-terminal amino group could also be reversibly protected or otherwise conjugated to the isothiocyanato group of the maleimidophenyl isothiocyanate (MPITC) linker with or without the DNA docking strand (DS) conjugated to the maleimido group of the linker. [0362] In one experiment, eleven different synthetic peptides differing only in the identity of the N-terminal amino acid (NTAA) were interrogated with IS1-AF488 and IS1-BODIPY-FL (FIG.4). A unique, differential response was detected for the tryptophan, arginine, phenylalanine, serine, and phospho-serine NTAA peptides. Furthermore, for the serine, arginine, and phenylalanine NTAA peptides, there were different responses measured for IS1-AF488 and IS1-BODIPY-FL. [0363] Embodiments at various assemblies were tested to determine if any component of the embodiment confounds the fluorescence signal. Each assembly was made following the current protocol in a separate well of an 8-well chambered slide. Fluorescence microscopy was performed and fluorescence intensity was collected from each sample with the same excitation and emission parameters. As shown in FIG.5, the fluorescence intensity was higher when the imaging strands were attached to the construct. This indicates that our collected fluorescence lifetime data collected is dominated by the fluorophores and suggests that other components of the construct do not produce significant fluorescence. [0364] To demonstrate the effect of fluorescence lifetime changes from different fluorophores conjugated to the same IS1 imaging strand, serine and phosphor-serine NTAA peptides were Filed: January 4, 2024 conjugated to the glass and used to compare the lifetimes from AF488, BODIPY-FL, BODIPY- TR, and TAMRA (Carboxytetramethylrhodamine). Within the experiment, the NTAA peptides remained unchanged while the IS1-conjugated fluorophore complex was cycled. Our results show that we can successfully and completely remove the fluorophores as when changing from a green fluorophore, such as AF488 or BODIPY-FL, to a red fluorophore, such as BODIPY-TR or TAMRA, we have minimal signal from the previous fluorophore in the green channel (not shown). Additionally, the fluorescence lifetime results suggest that some fluorophores may be more sensitive to some NTAAs than others (FIG.6). [0365] To test if alternative imaging strands change the fluorescence lifetime of fluorophores, three different synthetic peptides were interrogated sequentially with different DNA imaging strand (IS) and fluorophore conjugates (IS-fluorophore). These three peptides differed only in the identity of the N-terminal amino acid (NTAA): the first peptide contained an N-terminal glycine residue (G), the second peptide contained an N-terminal arginine residue (R), and the third peptide contained an N-terminal tryptophan residue (W). These peptides were sequentially interrogated with the following IS-fluorophore conjugates (the IS sequences are shown in (FIG.2): IS1-AF488, IS2-AF488, IS3-AF488, and IS3-BODIPY. For each different IS-fluorophore conjugate, the fluorescence lifetime was recorded. The data showed that both the sequence of the IS and the choice of fluorophore affected the measured fluorescence lifetime (FIG. 9). In brief, for the tryptophan NTAA peptide, the measured fluorescence lifetime was suppressed compared to the glycine and arginine NTAA peptides for each IS-AF488 combination. However, for IS3-BODIPY, the fluorescence lifetime measured with the tryptophan containing peptide was less suppressed than the other two peptides. Also, for IS1-AF488, a greater fluorescence lifetime was measured with the arginine NTAA peptide compared to the glycine NTAA peptide, but this trend was reversed for IS2-AF488 and IS3-AF488. [0366] To fully test the complete workflow, multiple synthetic peptides were interrogated with IS1- AF488 and IS1-BODIPY-FL before and after multiple Edman degradation cycles (FIGs.7 & 8). Prior to Edman degradation, with IS1-AF488 and IS1-BODIPY-FL, the arginine N-terminal amino acid (NTAA) peptide showed a higher normalized fluorescence lifetime measurement than the glycine NTAA peptide and the tryptophan NTAA peptide showed a lower fluorescence lifetime measurement than the glycine NTAA peptide. Additional measurements suggest that phenylalanine, tyrosine, histidine, and methionine have similar fluorescence lifetimes. Upon Edman degradation to remove the NTAA of each synthetic peptide and expose an N-terminal glycine residue for each peptide, the fluorescence lifetimes from IS1-AF488 and IS1-BODIPY-FL converged to similar values measured for the original glycine NTAA peptide as was theoretically predicted (FIGs.7A-7B). Additional experiments were performed where multiple Edman cycles Filed: January 4, 2024 were performed on synthetic peptides which contained tryptophan or arginine in the second (“N- 1”) or third (“N-2”) positions along the peptide. As shown in FIGs. 8A-8H, the fluorescence lifetimes from IS1-AF488 as well as IS1-BODIPY-FL fluctuated after each Edman degradation cycle suggesting that the new N-terminal amino acid, post-Edman is altering the fluorescence lifetime compared to the pre-Edman state [0367] These experiments demonstrated the validity of the workflow (illustrated in FIGs.1A-1G) by showing that the fluorescence lifetime measurements differ according to the choice of DNA imaging strand (IS) sequence, the choice of the fluorophore conjugated to the IS, and the identity of the N-terminal amino acid (NTAA) of the peptide. Furthermore, the Edman degradation proof of concept experiment showed that the workflow can be repeated for each subsequent amino acid within the polypeptide sequence until some or all of the amino acids have been interrogated. Methods [0368] Molecular Dynamics Simulations: Modeling and simulations were performed for predictions of optimizing linker length as well as functional dye-N-terminal amino acid combinations. The DNA docking strand (DS) and imaging strand (IS)-fluorophore oligonucleotides were modeled using a combination of homology modeling and energy minimization, followed by equilibration in all-atom molecular dynamics (MD) simulations in water. The Molecular Operating Environment (MOE) software was primarily used for the modeling and the initial energy minimization. [0369] All-atom MD simulations were performed using the GROningen MAchine for Computer Simulations (GROMACS-2018) on an Exacloud cluster at Oregon Health & Science University (Portland, OR). [0370] All-atom AMBER-type force field parameters were generated for the systems subject to all-atom MD simulations. AMBER tools were used to calculate partial charges at HF/6-31G (AM1- BCC) and use the General AMBER Force Field (GAFF) for bonded and van der Waals interactions, after ensuring the right protonation state for the molecules. [0371] MD simulations were carried out in an aqueous environment. The TIP3P water model was used to solvate the system in an aqueous environment with proper number of counterions (Na⁺ or Cl-) to ensure charge neutrality. A 3D periodic box was used to center the complex with at least 1.0 nm from the edge, accounting for >2 nm of solvent buffer. The 5 ns equilibration and 100 ns production runs were run in NPT ensemble, where the temperature was maintained at 300 K, and the pressure was maintained at 1 bar, using V-rescale thermostat and Parrinello–Rahman barostat, respectively. The MD simulations incorporated a leap-frog algorithm with a 2 fs time step Filed: January 4, 2024 to integrate the equations of motion. The long-ranged electrostatic interactions were calculated using particle mesh Ewald (PME) algorithm with a real space cutoff of 1.2 nm. LJ interactions were also truncated at 1.2 nm. LINCS algorithm was used to constrain the motion of hydrogen atoms bonded to heavy atoms. Co-ordinates of the protein molecule were stored every 1 ps for further analysis. To monitor the systems to reach equilibration, root-mean square deviations (RMSD) of the complex structures were calculated as a function of time. [0372] Surface Functionalization and Synthetic Peptide Attachment: Glass coverslips were etched with 6 mM KOH for 20 min at room temperature. The surface was then functionalized with a 1:1 solution of silane-PEG:silane-PEG-maleimide, to evenly distribute the maleimide- functionalized groups on the glass surface for addition of a peptide linker by a click chemical reaction. The silane linkers dissolved in a solution of 95% ethanol, 1% acetic acid, and the remainder was ultra-pure water with a final concentration of 3.2 mM of each linker. The glass coverslip was incubated at room temperature with the silane linker mixture for 30 min. After incubation, the coverslips were rinsed with ultrapure water. Synthetic peptides each containing a C-terminal cysteine residue were solubilized in ultrapure water at a final concentration of 1 µM, added to the glass surface, and incubated at room temperature for 4 h to covalently attach the sulfhydryl group of the cysteine side chain to the maleimido group on the functionalized surface. [0373] Synthesis of the Malemidophenyl Isothiocyanate (MPITC) Linker: To synthesize the MPITC linker, 0.2 mmol of the starting material (CAS registry number: 29753-26-2, IUPAC: 1-(4- aminophenyl)-1H-pyrrole-2,5-dione) was dissolved in 5 ml absolute ethanol in a round bottom flask. To the flask, a 10x molar equivalent of carbon disulfide (5 M in tetrahydrofuran) was added. Triethylamine was added in a 1:1 molar ratio to the starting material and the reaction mixture was stirred at room temperature for 30 min at 100 rpm using a stir bar. The flask was transferred to an ice bath. A catalytic amount (3 mol % of starting material) of 4-dimethylaminopyridine (DMAP) was dissolved in absolute ethanol. The DMAP solution and 98 mol % (relative to starting material) of di-tert-butyl decarbonate were added to the flask simultaneously and under stirring. The reaction mixture was incubated in the ice bath under stirring for 5 min then transferred to room temperature for overnight stirring at 200 rpm. Solvent was removed by rotary evaporation at 40 ºC and 175 mbar until visibly dry and then 40 ºC and 0 mbar for 5 min to yield a white crystalline solid. [0374] The product was confirmed using a nuclear magnetic resonance spectroscopy (NMR) and Fourier-transform infrared spectroscopy (FTIR). Proton and carbon NMR of the product in deuterated chloroform was performed on a 400 MHz Avance NEO NanoBay spectrometer (Bruker, Inc.). FTIR was performed on a Nicolet iS5 KBR window FTIR spectrometer (Thermo Fisher Scientific, Inc.) with an iD7 anti-reflectance diamond crystal attenuated total reflectance Filed: January 4, 2024 (ATR) module. Onto the ATR crystal, 2 µl of 7.4 mM MPITC or 2 µl of 7.4 mM starting material in absolute ethanol was directly added. Each sample was dried under a stream of clean dry air, scanned 256 times at 2 cm^-1 resolution from 4000 to 400 cm^-1. [0375] Conjugation of a Peptide to a DNA Docking Strand using the MPITC Linker: The N-terminal amine of a peptide was first covalently attached to the isothiocyanato group of MPITC (FIG.3). MPITC at a concentration of 7.46 mM in absolute ethanol was incubated on the peptide- functionalized glass coverslip for 5 h to complete the conjugation. The MPITC-peptide functionalized glass coverslip was washed with ultrapure water. Next, a DNA docking strand (DS) oligonucleotide with a 3’ (3-mercaptopropyl)phosphate was covalently attached to the maleimido group of MPITC (FIG.3). A 1 nmol quantity of a 100 µM DS solution was diluted to 10 µM in DNA buffer with 1 µl tris(2-carboxyethyl)phosphine (TCEP) to reduce the 3’ modification and expose a free sulfhydryl group. This mixture was added to the MPITC-peptide functionalized surface and incubated at room temperature for 3 h to yield a DS-MPITC-peptide functionalized surface. [0376] Preparation of DNA Imaging Strand-Fluorophore Conjugates: To conjugate each fluorophore to each DNA imaging strand (IS), a maleimide-functionalized fluorophore was dissolved at a concentration of approximately 20 mM in pure DMSO and a IS with a 5’ (6- aminohexyl)phosphate modification was dissolved to 1 mM in ultrapure, nuclease free water. For each fluorophore and IS combination, the fluorophore and IS are combined together at 1:10 and 1:20 dilutions, respectively in ultrapure water containing 10% 1 M NaHCO3, with a pH of approximately 8.0. The fluorophore and IS were reacted at room temperature with 500 rpm shaking on a ThermoMixer (Eppendorf GmbH) to form a covalent linkage between the 5’ amino group of the IS and the maleimido group of the fluorophore (FIG.3). [0377] To purify with IS-fluorophore conjugate from excess fluorophore, the solution was mixed 1:1 with 3 M sodium acetate. This solution was diluted 1:5 in absolute ethanol and incubated at - 80 ºC overnight to precipitate the IS-fluorophore conjugate. The precipitated IS-fluorophore conjugate was pelleted by centrifugation at approximately 20000 × g for 30 min at 2 ºC, the supernatant was removed, and then the pellet was resuspended in -20 ºC absolute ethanol; these centrifugal washing steps were repeated three times. The pellet was resuspended in 50 µl ultrapure water and diluted 1:1 with 3 M sodium acetate, absolute ethanol was added to a concentration of 80% v/v, and this solution was incubated overnight at -80 ºC. The precipitated IS-fluorophore conjugate was pelleted and resuspended three times as described then the pellet was lyophilized for 10 min or until dry. The lyophilized conjugate was resuspended in ultrapure water. Absorbance at 260 nm was measured to determined concentration of the IS and absorbance was measured to calculate dye concentration based on the absorptive properties and extinction coefficient of the fluorophore. IS-fluorophore conjugates were stored at -20 ºC. Filed: January 4, 2024 [0378] DNA Docking Strand:Imaging Strand Hybridization and Imaging Strand Removal: To anneal the imaging strand (IS)-fluorophore conjugate to the peptide-conjugated DNA docking strand (DS), 1 nmol of IS-fluorophore conjugate was at a concentration of 10 µM in DNA buffer was added to the DS-MPITC-peptide functionalized surface and incubated at RT for 1–5 min for hybridization to occur. After hybridization and thorough washing with 10 mM HEPES buffer to remove excess, unhybridized IS-fluorophore, fluorescence lifetime measurements were taken. [0379] To perform multiple interrogations of the N-terminal amino acid (NTAA), the IS-fluorophore conjugate can be removed and an IS-fluorophore conjugate with a different oligonucleotide sequence and/or fluorophore can be annealed to the DS for further fluorescence lifetime measurements. Removal of an IS-fluorophore conjugate from the DS was achieved through incubation with a chemical denaturant, such at 8 M urea for 1–5 min, followed by thorough washing with 10 mM HEPES buffer to remove the freed IS-fluorophore conjugate. [0380] Fluorescence Lifetime Measurement: Fluorescence lifetime imaging (FLIM) of all synthetic peptides was performed on a Zeiss LSM 880 scanning confocal microscope equipped with a Chameleon Ti:Sapphire (Coherent) multi-photon source operating at a pulse repetition frequency of 80 MHz. Multi-photon excitation of the sample was achieved by scanning the excitation beam over a 144 x 144 µm² area at 1% power. Single-photon fluorescence events were captured on a Big.2 gallium arsenide phosphide photomultiplier tube (GaAsP-PMT) after passing through the 1.4 NA 63x magnified objective and 640 nm long-pass filter to construct a 512 x 512 image with 290 nm pixels. Areas were scanned approximately 100 times to fill the fluorescence lifetime distribution for each pixel. Time-correlated single photon counting (TCSPC) was performed with Becker & Hickel TCSPC electronics and SPCM software (B&H). [0381] FLIM data analysis was performed with FLIMfit (accessible online at flimfit.org/), an open- source software tool, using Matlab Compiler Runtime R2016b. To characterize the system, an instrument response function (IRF) was determined via second harmonic generation imaging of dried urea crystals on a microscope slide. Using a nonlinear least squares method, the lifetime measurements from the fluorophores were fitted via the following expression at each pixel within the image: ^ ^ [0382] where ^_^^^^^^^^^^ is from ambient light and detector noise. Ai and τi are the amplitude and lifetime from the exponential fits, respectively. Fitting of the lifetime curve was assessed via evaluating the mean χ². A χ² of less than 1.2 and Filed: January 4, 2024 greater than 0.88 was determined as a good fit of the decay. From the fit equation, a mean lifetime was calculated: ^ = ^{∑^} ^_^^ ^_^^_{^ ^^^^} ∑^{^} ^_^^ ^_^ [0383] where αi is the fractional i within the exponential decay fit, which the sum of fractional amplitudes from 1. The calculated mean lifetime was averaged over all pixels of the image for samples containing a known single synthetic peptide. [0384] Edman Degradation to Remove N-Terminal Amino Acid (NTAA): Edman degradation was performed to remove the N-terminal amino acid (NTAA) and to expose the N-terminal amine of the next amino acid in the polypeptide chain. Aqueous TFA at a concentration of 0.5% v/v (pH 2) was added to the DS-MPITC-peptide or IS:DS-MPITC-peptide functionalized glass surface and incubated at 55ºC for 40 min. The surface was then washed with 10 mM HEPES buffer extensively to discard all removed NTAA-linked oligonucleotide and prepare for the next reaction with the MPITC linker. [0385] Microfluidic Chip Preparation: For some experiments, a microfluidic chip was assembled around the peptide-functionalized glass coverslip and subsequent chemical reactions were performed within the microfluidic chip. [0386] The microfluidic contains polydimethylsiloxane (PDMS) guides for the inlet and outlet microfluidic tubing. To produce the PDMS for these guides, a Sylgard 184 Silicone Elastomer (Dow, Inc.) was used. The components were combined according to the kit instructions (10:1 dimethylsiloxane to siloxanes and silicones), mixed thoroughly, and then bubbles were removed under vacuum. The PDMS mixture was poured onto a clean silicon wafer and baked at 80ºC for 90 min in an oven. The cured PDMS was cut to size with a scalpel and tube channels were made using a 1 mm diameter biopsy punch. The punched PDMS was washed thoroughly in IPA and ultrapure water. Holes to accommodate microfluidic tubing were drilled into a glass slide using a diamond-reinforced bit. The punched PDMS and drilled glass slide were treated with oxygen plasma with argon carrier gas. The plasma-treated surfaces of the punched PDMS and drilled glass slide were aligned with an inspection microscope and then baked for 30 min at 100-105ºC on a hotplate or in an oven to adhere the plasma-treated surfaces. [0387] Next, a double-sided adhesive, such as 127 µm-thick acrylic adhesive 200MP Model 468MP (3M Co.), was cut to the desired geometry, such as an oval, and applied around the drilled holes of the glass slide. Then, a peptide functionalized glass coverslip prepared as described herein, was adhered to the tape to form an enclosed microchannel. For fluid handling, thin-walled Teflon (PTFE) tubing, such as TT-26 with inner diameter 0.018 in and 0.009 in wall thickness (Weico Wire & Cable, Inc.), was inserted through the PDMS guides, through the drilled holes in Filed: January 4, 2024 the glass slide, and into the microchannel of the microfluidic chip. All chemical reactions can be performed via injection of reaction components into the microchannel via syringe. Example 2: Identification of Amino Acids with Machine Learning Algorithm(s) [0388] Empirical and Monte-Carlo simulated time-correlated single photon counting (TCSPC) lifetime data can be used to train a convolutional neural network (CNN) for amino acid calling. To do so, fluorescence lifetimes (such as those generated using methods as in Example 1) can be abstracted into a pixel array of intensities weighted by the average lifetime of single molecules. The resulting image corresponds to a unique amino acid fingerprint that can be interrogated by the deep learning network. [0389] The cyclic nature of the described approach allows us to probe a single residue and construct a large M × N matrix of intensities where the fields M and N are “fluorophores” and “imaging strands”, respectively. To minimize residual error in fitting, these conditions can be combined with other spectroscopic techniques to construct an n-dimensional tensor for input into the CNN. For example, the CNN can receive a 3-dimensional tensor with the fields M, N, and L; where L is an average value derived from the fluorescence autocorrelation function. Example 3 [0390] This example demonstrates that use of alternative imaging strand designs can be used to enhance flexibility in the provided peptide sequencing methods and systems. [0391] Single stranded DNA (ssDNA) docking and imaging strand sequences and schematic arrangement are shown in FIG.2. The ssDNA imaging strands (IS) can be modified in a variety of ways, such as inclusion of modified or non-natural nucleotides, inclusion of a 5’ IS overhang relative to the docking strand (DS), or inclusion of a 5’ IS underhang relative to the DS. These variables adjust the spatial position and/or degrees of freedom of the attached fluorophore, and thereby modulate interactions with the NTAA side chain, which modulates the measured fluorescence lifetime. This enables identification of each NTAA. [0392] Data obtained using the same peptides, but with different Imaging Strands (IS1 (SEQ ID NO: 2), IS2 (SEQ ID NO: 3), and IS3 (SEQ ID NO: 4)) and fluorophores (Alexa Fluor® 488 in the top panel; BODIPY in the bottom), are shown in FIG.9. Data are normalized to the fluorescence lifetime of the respective free fluorescent dye. IS1 and IS2 have higher melting temperatures, and IS3 has single base overhang. The data illustrates that using alternative IS designs can be used to enhance flexibility in peptide sequencing analysis. Filed: January 4, 2024 Example 4 [0393] This Example demonstrates that the herein provided peptide analysis methods are useful to generate unique peptide identification fingerprints. [0394] Fluorescence lifetimes from the workflow were plotted for AF488 and BODIPY when proximal to W, F, Y, G, H, M, R on the N-terminus of peptides (FIG.10A). Marker radius represents standard deviation of the bulk distribution of lifetimes for each dye-AA measurement. Lines for W and G were plotted to demonstrate unique fingerprinting between each amino acid. [0395] Simulated 2-dimensional gaussian distributions of dye-AA lifetimes for AF488 and BODIPY were generated using the empirically derived mean and standard deviation of fluorescence lifetime measurements observed in the workflow. The two-dimensional distributions in FIG.10B were plotted as scatter-histograms to demonstrate how amino acids may be called (identified) using multiple dyes. Alternative fluorescent lifetime fingerprinting highlights prediction of different N-terminal amino acids with the use of two separate fluorophores attached to IS1. Point spread fluorescence lifetimes from AF488 and BODIPY-FL with tryptophan, glycine, and arginine are depicted. As shown, it may be challenging to predict differences in fluorescence lifetimes between three amino acids with only one dye; however, with using two dyes, separation of species can be observed. Thus, FIG.10B illustrates that fluorophore cycling creates a robust amino acid fingerprint. [0396] Complex multivariate results increase accuracy of neural network prediction. Dye-AA lifetimes generated in the workflow were used to generate 3-dimensional arrays (FIG. 10C). Intensity of the pixel blocks represent lifetimes, the Y-axis represents dye measurements with differing imaging strands, and the X-axis represents unknown amino acids. These image arrays produce a fingerprint that can be used in a convolutional neural network for amino acid prediction and sequencing. Fluorescent lifetime fingerprint of different N-terminal amino acids is illustrated, with a theoretical neural network approach for the identification of the amino acids. Example 5 [0397] This Example provides evidence that provided methods can also operate where the free, N-terminus of the peptide is linked to a modified nucleotide within the DS oligonucleotide sequence rather than at (or near) a terminal end of the DS oligonucleotide. As illustrated in this example, the fluorophore was linked to a modified nucleotide within the IS oligonucleotide sequence rather than at (or near) to the terminal end of the IS oligonucleotide. [0398] FIG.11 illustrates an alternative embodiment of the provided peptide sequencing system, using a DNA major groove design. This embodiment provides additional tunable control over the interaction between the fluorescent dye and structural components of the DNA docking Filed: January 4, 2024 strand:imaging strand (DS:IS) complex. In contrast to the embodiments wherein interaction is between the fluorescent dye and the nucleobases of the blunt end of the DS:IS complex, in this alternative embodiment the fluorescent dye is conjugated internally within the IS and therefore cannot access the blunt end of the complex. This has been verified with molecular dynamics simulations in silico. The immobilized peptide is conjugated to a modified nucleotide within the DS (that is, not immediately proximal to either end of the DS) and the fluorophore is conjugated to a modified nucleotide within the IS (that is, not immediately proximal to either end of the IS). In silico molecular dynamics simulations have shown that for this embodiment, the peptide and fluorophore can be positioned within the major groove of the double stranded DNA to maximize the interaction between the fluorophore to the N-terminal amino acid sidechain of the peptide. FIG.11 shows one such configuration generated by MD simulations that were described in the methods section. In this configuration, the N-terminus of the peptide is attached to a thiol modified dT nucleobase on the DS oligonucleotide using the maleimide phenyl isothiocyanate bifunctional linker. The NHS fluorophore is attached to an amine modified dT nucleobase on the IS oligonucleotide.3D modeling revealed that attachments of the peptide and the fluorophore on the fifth position of the nucleobase position them on the major groove of the DNA double helix, and to the 3’ side of their respective oligonucleotides. For the linkers described here, MD simulations show that when the fluorophore and the peptides are kept four to six bases apart, their interaction is maximized. Furthermore, keeping the fluorophore away from the terminus of the oligonucleotide minimizes any interaction of the fluorophore with the nucleobases of the DS and IS oligonucleotides. Example 6 [0399] This Example demonstrates variation in fluorescence lifetime from fluorophores conjugated to IS1. [0400] Using methods essentially similar to those described in Example 1, various fluorophores were conjugated to IS1 and each IS was suspended in water. Fluorescence lifetimes were measured for each IS-fluorophore conjugate (FIG. 12). The longer-lifetime fluorophores demonstrated greater differences in lifetime with KU560-6 possessing a significantly higher lifetime among the KU dyes. These greater differences in lifetime may increase sensitivity when measuring lifetime differences among the AAs with little differences as measured by AF488 and BODIPY-FL. Filed: January 4, 2024 Example 7 [0401] This Example demonstrates that the herein provided peptide analysis methods are useful to generate unique peptide identification fingerprints with the use of longer lifetime KU™ Dyes. [0402] FIG. 13 is a bar graph that illustrates the various fluorescence lifetimes measured from various imaging strands containing long-lifetime fluorophores, KU530-6 and KU530-R-4 to identify the N-terminal amino acids. [0403] To screen the peptides, fluorescence lifetimes were measured from long-lifetime fluorophores, KU530-6 and KU530-R-4 when proximal to W, F, Y, G, H, M, Q, E, S, R on the N- terminus within the established workflow (FIG. 13). When comparing the lifetimes reported by each dye for the particular AA, there were measured differences for W, Y, and H. Additionally, KU530-6 showed significant changes in lifetime between several amino acids. When combined with the previously tested AF488 and BODIPY-FL, the additional dyes contribute to defining a characteristic lifetime fingerprint for each amino acid that can be used to distinguish between terminal AAs. Example 8 [0404] This Example demonstrates that the herein provided peptide analysis methods are useful to identify post-translational modifications on NTAAs. [0405] Fluorescence lifetimes measured from imaging strands (IS1) conjugated with Alexa Fluor 488, BODIPY-FL, or KU530-6 that were hybridized to surface-bound peptides that possessed common post-translational modifications of either phosphorylation on serine or acetylation of lysine at the N-terminus. There were no measured differences in lifetimes measured from AF488 with acetylated lysine and phosphorylated serine; however, KU530-6 reported a different lifetime when comparing the two PTMs (FIG.14). This highlights the potential of using the longer lifetime dyes to discern some common post-translationally modified amino acids. When comparing the lifetimes of phosphorylated serine, BODIPY-FL reported a significantly higher lifetime than AF488, demonstrating that the use of multiple dyes may help elucidate the PTM. [*, p<0.05; One-way ANOVA with Tukey Post hoc; N=multiple fields of view within 3 samples]. This demonstrates that each dye has a different interaction and hence lifetime between various amino acids which can ultimately be used to sequence the peptide. [0406] To further elucidate the molecule contributing to the difference in lifetime, fluorescence lifetimes were collected from IS1 conjugated with AF488 in control samples of SGG as well as post-translationally modified serine (PhosSGG). After FLIM was performed, the IS1-AF488 was removed, and a phosphatase was added to the PTM samples to remove the phosphorylated group on the modified serine (PhosSGG). IS1-AF488 was reintroduced to hybridize with the DS Filed: January 4, 2024 and FLIM conducted. In FIG.15, the bar graph depicts lifetimes measured from unmodified serine (SGG), phosphorylated serine PTM (PhosSGG), and the dephosphorylated PTM (DePhosSGG). Results show that the lifetimes reported from dephosphorylated serine (DePhos) were not significantly different to unmodified controls (SGG) and phosphorylated serine PTM was significantly lower than the control [*, p<0.05; One-way ANOVA with Tukey Post hoc; N = multiple regions within 3 samples]. This demonstrates that the phosphorylation on the modified serine was altering the lifetime. Additionally, AF488-conjugated to IS1 may be a good candidate to detect differences in PTMs compared to unaltered peptides. Example 9 [0407] This Example provides a visual depiction of the sequencing data to interpret the data as well as determine variations in the lifetime data collected and further demonstrate the capability of this sequencing approach. [0408] Normalized lifetime “heatmap” from sequenced data as reported from four separate fluorophore-conjugated imaging strands with the peptide screening experiments (FIGs.4 and 13). Each data was normalized to the measured lifetime of GGGS for each fluorophore-conjugated IS and patterned based on the corresponding range of normalized lifetimes. Following the established workflow, peptides which terminated in G, W, F, Y, H, M, Q, E, S, or R were separately adhered to a solid substrate where each group were in separate wells. After DS attachment, IS1- AF488 was hybridized, and lifetime data was collected for each separate peptide sequence. Dehybridization of IS1-AF488 followed by hybridization of a different IS1-fluorophore conjugate was performed. Lifetimes were collected. This cycle was repeated for all IS1-fluorophore conjugates listed in FIGs. 4 and 13 (BODIPY-FL, KU530-6, and KU530-R-4). The collected lifetime information from each peptide and fluorophore were normalized to GGGS for each fluorophore. Data was compiled and presented in a heatmap based on their normalized lifetimes (FIG.16). Each amino acid results in a distinct pattern of lifetimes across the four dyes, with no two showing the same response pattern. This demonstrates that by using additional dyes, even amino acids with similar lifetimes for a given fluorophore-IS combination can be resolved. Post- translational modifications on the terminal amino acid were tested and mapped similarly for phosphorylated serine and acetylated lysine with AF488, BODIPY-FL, and KU530-6 conjugated to IS1. The normalized lifetimes with each fluorophore are similarly characteristic to the modified amino acids and do not coincide with any of the other characteristic responses, suggesting that PTMs may also be successfully identified using this method. Filed: January 4, 2024 Example 10 [0409] This Example determines the effect of certain amino acids further along the peptide on the measured fluorescence lifetime. Amino acids at the second (“N-1”) and third (“N-2”) position from the N-terminus were modified with glycine surrounding the amino acid. Lifetimes were collected with standard workflow. [0410] Tryptophan and arginine were incorporated at the second (“N-1”) and third (“N-2”) positions along otherwise identical peptides to determine whether amino acids at N-1 or N-2 affect fluorescence lifetime (FIG.17). AF488 was conjugated to IS1 and used with each peptide. When compared to the control, GGGS, the glycine-terminated peptides with W or R at N-1 or N-2 did not show a statistically significant difference in lifetime, demonstrating that the AF488 is sensitive to only the terminal AA of the peptide. [N= multiple fields of view within 3 samples]. However, a slight increase in lifetime with R at N-2 was observed, which suggests that IS1-AF488 may be sensitive to arginine at another position further along the peptide sequence. [0411] Tryptophan and arginine were incorporated at the second (“N-1”) and third (“N-2”) positions along otherwise identical peptides to determine whether amino acids at N-1 or N-2 affect fluorescence lifetime (FIG.18). BODIPY-FL was conjugated to IS1 and used with each peptide. When compared to the control, GGGS, the glycine-terminated peptides with W or R at N-1 or N- 2 did not show a statistically significant difference in lifetime, demonstrating that the BODIPY-FL is sensitive to only the terminal AA of the peptide. However, there was a slight decrease in lifetime with R at N-1 and an increase in lifetime with R at N-2 compared to GGGS, which suggests that IS1-BODIPY-FL may be sensitive to arginine at others position further along the peptide sequence. This IS1-fluorophore could be used to further sequence amino acids along the peptide sequence without the use of Edman degradation, with the aid from trained machine models. [0412] In FIG.19, fluorescence lifetimes were measured from imaging strands conjugated with KU530-6 with peptides that possessed a tryptophan or arginine at the second and third positions along the peptide to determine if amino acids at N-1 or N-2 affect the lifetime of the fluorophore. KU530-6 demonstrated minimal sensitivity to amino acids further along the sequence, ultimately highlighting the specificity of KU530-6 in interacting with the terminal amino acid and manipulating the lifetime of the dye. [*, p<0.05; ***, p<0.005; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. Example 11 [0413] This Example demonstrates the complete workflow of the embodiment. This includes binding the construct to the glass substrate, performing FLIM, cycling imaging strands, as well as cyclic Edman degradation to further determine the sequence of the peptide. This Example focuses Filed: January 4, 2024 on the Edman degradation process and resulting lifetime changes based on the new terminal amino acid. [0414] Peptides that possessed sequences of different NTAAs (G, W, R, F, Y, H, and M) and identical remaining sequences were adhered to a glass substrate following the established workflow. Imaging strands (IS1) containing KU530-6 were hybridized to the conjugated DS on the peptide. FLIM was performed for each group and then Edman degradation was performed the remove the NTAA as well as the DS, linker and IS. A new linker was attached following DS conjugation. A new IS1-KU530-6 was hybridized and FLIM was performed for each group (Edman cleaved amino acid shown in parentheses). Data collected was normalized to GGGS (FIG.20). There were measured lifetime differences with tryptophan and arginine as the terminal amino acid compared to the Edman cleaved counterpart, suggesting that Edman degradation went to completion as the lifetime stabilized to values similar to GGGS. [**, p<0.01; One-way ANOVA, Tukey Post hoc; N= multiple fields of view within 3 samples]. Additionally, after the first Edman degradation cycle for each group, the lifetimes were similar among groups which demonstrates the capability to perform Edman degradation cycles with the existing workflow as well as IS1- KU530-6 reporting similar values with each group. [0415] Sequential analysis of amino acids from longer peptides were performed to demonstrate viability of the full workflow illustrated in FIGs. 1A-1G. A solid glass surface was etched with potassium hydroxide to possess -OH groups on the surface. Silane-PEG-maleimide was added to bind to the silane to the -OH groups. Synthetic peptides with sequences WGRSGGDC, RGWSGGDC, or WRGSGGSDC were tested immobilized to the glass substrate via cysteine- maleimide reactions. Each peptide was conjugated with the MPITC linker before covalently linking the MPITC linker to the DNA docking strand (DS) oligonucleotide. AF488 was separately conjugated to IS1, then added to the DS-MPITC-peptide and annealed to hybridize the DS and IS. Fluorescence lifetime was measured for the fluorophore. IS1-AF488 was then dehybridized, IS1-BODIPY was added to hybridize to the DS. FLIM was performed for that IS. Each IS1- conjugated fluorophore was cycled until all four fluorophores was used. Edman degradation was then performed to cleave the N-terminal AA. The new NTAA was then exposed to the same process of linker and DS attachment, then hybridization to the IS-fluorophore construct, FLIM measurement, IS-fluorophore cycling and Edman degradation. [0416] This complete process was completed three times and the sequential lifetime measurements are presented in FIGs. 21A-21C. The same workflow was completed using BODIPY-FL (FIGs.22A-22C), KU530-6 (FIGs.23A-23C), and KU530-R-4 (FIGs.24A-24C). The data from these experiments is summarized in FIG. 25 as a heatmap, where the fluorescence lifetime for each analysis step is normalized to GGGS. Each of the peptides has a unique series Filed: January 4, 2024 of lifetimes for each dye, indicating that the full workflow can be successfully completed with the existing fluorophores to sequence the full peptide. Additionally, results show that amino acids in the N-1 and N-2 position may also influence fluorescence lifetime for each dye; however, using machine learning algorithms will help further elucidate the differences and ultimately be used to sequence the full peptide. [0417] In FIG.25, the measured lifetimes as reported from FIGs.23A-24C were normalized and combined to create a lifetime “heatmap” depicting the sequenced data from four separate fluorophore-conjugated imaging strands. The heatmap shows the mean lifetime value that was normalized to GGGS for each respective fluorophore and patterned based on the corresponding range of normalized lifetimes. Each amino acid resulted in a distinct pattern of lifetimes across the four dyes as well as each peptide sequence. This demonstrates that by using the chosen dyes, amino acids with similar lifetimes for a given fluorophore-IS combination can be resolved. Furthermore, this heatmap highlights the sensitivity of some fluorophores to amino acids at N-1 and N-2 such as IS1-AF488 and WGRSGGSDC (SEQ ID NO: 5). After three Edman degradation cycles, the sequence of all peptides contained the same sequence (XXX)SGGSDC (SEQ ID NO: 8). Measured lifetimes from this sequence were not significantly different among the peptides for BODIPY-FL, KU530-6, and KU530-R-4; however, the reported lifetime from (RGW)SGGSDC (SEQ ID NO: 6) was significantly lower than (WGR)SGGSDC (SEQ ID NO: 5) and (WRG)SGGSDC (SEQ ID NO: 7) for IS1-AF488. This suggests that the samples were not completely washed as the following BODIPY-FL-conjugated IS1 did not possess differing lifetimes among all the peptide sequences. These data demonstrate the full workflow in sequencing peptides up to 4 amino acids within a sequence using 4 imaging strands and producing varying lifetimes which can be further elucidates with machine learning algorithms. Example 12 [0418] This Example highlights the variability in measured lifetimes with various longer lifetime dyes with the current workflow. Selection of the candidates to use for most of the study was performed. [0419] In FIG. 26, initial screening was performed using commercially available dyes which possess longer lifetimes, KU530-6, KU530-R-4, KU560-6, KU560-R-4 that were conjugated to IS1 and hybridized to DS that were covalently attached to surface-immobilized peptides containing different NTAAs, tryptophan, arginine, and glycine. Longer lifetimes were reported compared to AF488 and BODIPY-FL. The three peptides chosen had significant different lifetimes for KU530-6. Arginine had a significantly higher lifetime than WGG or KU560-6. [*, p<0.05; ***, p<0.005; ****, p<0.001; One-way ANOVA with Tukey post hoc; N=multiple fields of view within 3 Filed: January 4, 2024 samples] KU530-6 was chosen to be used for this embodiment due to the demonstrated sensitivity to the amino acids. KU530-R-4 was chosen as it had the same fluorophore to KU530- 6 but possessed a rigid linking group on it which may create different interactions with the amino acids. It appeared to report an inverse relationship of lifetimes compared to KU530-6. (XV) Select References [0420] US Patent No.8,373,115 Method and apparatus for identifying proteins in mixtures [0421] US Patent No.10,545,153 Single molecule peptide sequencing [0422] US Patent No.10,852,230 Molecules and methods for iterative polypeptide analysis and processing [0423] US Patent No.11,001,875 Methods for nucleic acid sequencing [0424] US Patent No.11,162,952 Single molecule peptide sequencing [0425] US Patent No.11,268,963 Protein sequencing methods and reagents [0426] US Application Publication No. 2014/0273004 Molecules and methods for iterative polypeptide analysis and processing [0427] US Application Publication No.2019/0145982 Macromolecule analysis employing nucleic acid encoding [0428] US Application Publication No. 2021/0396762 Methods for peptide analysis employing multi-component detection agent and related kits [0429] International Patent Publication No. WO 2010/065531 Single molecule protein screening [0430] International Patent Publication No. WO 2016069124 Improved single molecule peptide sequencing [0431] Anju et al., ACS Omega 4:12357-12365, 2019 [0432] Swaminathan et al., Nat. Biotechnol. DOI: 10.1038/nbt.4278.2018 [0433] Timp & Timp, Sci Adv.6: eaax8978 (16 pages), 2020 (XVI) Closing Paragraphs [0434] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified. The transition phrase “consisting Filed: January 4, 2024 essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. [0435] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value. [0436] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [0437] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No Filed: January 4, 2024 language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0438] Throughout this disclosure, various aspects or variables are presented in a range format. It is understood that the description in range format is merely for convenience and brevity; this is not to be construed as an inflexible limitation on the scope. Accordingly, the description of a range will be understood to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 is intended to be viewed as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 7, from 2 to 4, from 2 to 6, from 3 to 6, and so forth, as well as individual numbers within that range, such as specifically 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range, and is only limited to integer amounts where context requires that it do so. [0439] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0440] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [0441] Furthermore, numerous references have been made to patents, printed publications, journal articles, other written text, and web site content throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching(s), as of the filing date of the first application in the priority chain in which the specific reference was included. For instance, with regard to chemical compounds, nucleic acid, and amino acids sequences referenced herein that are available in a public database, the information in the database entry is incorporated herein by Filed: January 4, 2024 reference as of the date of an application in the priority chain in which the database identifier for that compound or sequence was first included in the text. [0442] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described. [0443] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. [0444] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2^nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and/or A Dictionary of Chemistry, 8^th Edition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

Filed: January 4, 2024 LISTING OF CLAIMS What is claimed is: 1. A method for identifying a terminal amino acid (TAA) of a peptide having a N-terminal amino acid (NTAA) and a C-terminal amino acid (CTAA), the method comprising: binding either the NTAA of the peptide or the CTAA of the peptide to a solid surface to produce a bound TAA; attaching to the non-bound TAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial TAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. 2. A method of sequencing a peptide having an initial terminal amino acid (TAA), comprising: interrogation of the initial TAA using a single-stranded DNA (ssDNA) docking strand (DS) attached to the initial TAA and a ssDNA imaging strand (IS) to which a signal molecule is conjugated, to produce a measurement of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide; wherein the IS and DS are at least partially complementary in sequence. 3. The method of claim 2, further comprising: sequential interrogation of the initial TAA using a library of at least two different combinations of a single-stranded DNA (ssDNA) docking strand (DS) and a ssDNA imaging strand (IS), where a signal molecule is conjugated to the IS, to produce a set of spectral characteristic data having a characteristic fingerprint for the initial TAA of the peptide. 4. The method of claim 2, wherein the initial TAA is: the N-terminal amino acid (NTAA) of the peptide; or the C-terminal amino acid (CTAA) of the peptide. Filed: January 4, 2024 5. The method of claim 2, wherein the method is carried out in parallel on a plurality of peptides. 6. The method of claim 2, wherein the signal molecule comprises a fluorophore, and the spectral characteristic comprises a measure of fluorescence. 7. The method of claim 6, wherein the spectral characteristic comprises fluorescence lifetime. 8. A method of sequencing a peptide having an initial N-terminal amino acid (NTAA) and a C-terminal amino acid (CTAA), the method comprising: sequential interrogation of the initial NTAA using a library of at least two different combinations of a ssDNA docking strand (DS) and a ssDNA imaging strand (IS), where a fluorophore is conjugated to the IS, to produce a set of fluorescence lifetime data having a characteristic measurement for each combination of DS, IS, and fluorophore, wherein each pair of IS and DS is at least partially complementary in sequence. 9. The method of any one of claims 1-8, wherein the DS is a universal DS. 10. The method of any one of claims 1-8, wherein the interrogation or the sequential interrogation comprises detecting and/or measuring interaction between fluorophore and amino acid sidechain at or near the CTAA or the NTAA by detecting fluorescence lifetime data for each of a plurality of IS / DS pairs in the library. 11. The method of claim 10, wherein detecting or measuring the interaction comprises obtaining fluorescence lifetime imaging (FLIM) single-molecule fluorescence measurements for each of a plurality of IS / DS pairs in the library. 12. The method of claim 1 or claim 4, further comprising removing the initial CTAA or NTAA of the peptide by an Edman degradation reaction, enzymatic digestion, or a similar process. 13. The method of any one of claims 1-8, wherein the method is repeated for at least two subsequent amino acids in the peptide to produce a matrix of fluorescence lifetime data. Filed: January 4, 2024 14. The method of claim 13, wherein the method is completed for each subsequent amino acid in the peptide to produce a matrix of fluorescence lifetime data. 15. The method of claim 13, wherein the fluorescence lifetime data is input into a machine learning algorithm to reconstruct a polypeptide sequence. 16. The method of claim 14, wherein the fluorescence lifetime data is input into a machine learning algorithm to reconstruct a polypeptide sequence. 17. The method of claim 3 or claim 8, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary so that spatial positioning and/or degrees of freedom of the attached fluorophore varies, in order to modulate interactions with the CTAA or NTAA side chain, and thereby modulate the measured fluorescence lifetime. 18. The method of claim 17, wherein the library of ISs includes a plurality of ssDNA oligonucleotides that vary by one or more of: inclusion of a modified nucleotide, inclusion of a non-natural nucleotide, inclusion of a 5’ IS overhang relative to the cognate DS, or inclusion of a 5’ IS underhang relative to the cognate DS. 19. The method of claim 10, wherein the interaction between the CTAA or NTAA is further influenced by one or more of DS position, degrees of freedom, or another variable described herein. 20. The method of any one of claim 1, claim 6, or claim 8, wherein the fluorophore comprises Alexa Fluor® 488 (AF488), BODIPY-FL, BODIPY-TR, TAMRA, or a KU dye. 21. The method of claim 20, wherein the fluorophore is conjugated at an end of the IS. 22. The method of any one of claims 1-8, wherein: the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide within the IS; or the peptide is conjugated to a modified nucleotide at or near an end of the IS and the fluorophore is conjugated to a modified nucleotide within the IS; or Filed: January 4, 2024 the peptide is conjugated to a modified nucleotide within the DS and the fluorophore is conjugated to a modified nucleotide at or near an end of the IS; or the peptide is conjugated to a modified nucleotide at or near an end of the DS and the fluorophore is conjugated to a modified nucleotide at or near an end of the IS. 23. The method of claim 12, wherein removing a CTAA or a NTAA is performed under conditions such that the remaining peptide has a new terminal amino acid available for another cycle of analysis. 24. The method of any one of claim 1-8, wherein the peptide or each peptide is immobilized on a solid support. 25. A database containing the matrix of fluorescence lifetime data of claim 14. 26. A method for identifying a N-terminal amino acid (NTAA) of a peptide having a NTAA and a C-terminal amino acid (CTAA), the method comprising: binding the CTAA of the peptide to a solid surface; attaching to the NTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. 27. A method for identifying a C-terminal amino acid (CTAA) of a peptide having a CTAA and a N-terminal amino acid (NTAA), the method comprising: binding the NTAA of the peptide to a solid surface; attaching to the CTAA of the peptide a ssDNA docking strand (DS); hybridizing to the DS a first ssDNA imaging strand (IS), which first IS includes a first fluorophore; detecting fluorescence lifetime data for the first fluorophore; dissociating the first IS from the DS; Filed: January 4, 2024 hybridizing to the DS a second ssDNA IS, which second IS includes a second fluorophore; detecting fluorescence lifetime data for the second fluorophore; and identifying the initial NTAA of the peptide based on the detected fluorescence lifetime data of the first and the second fluorophore. 28. The method of claim 26 or claim 27, further comprising: cleaving the initial TAA from the peptide, to leave a next TAA of the peptide. 29. The method of claim 28, wherein cleaving the initial NTAA comprises an Edman degradation reaction, Edman degradation enzyme reaction, or a similar process. 30. The method of claim 27 or claim 27, comprising repeating the method a plurality of times to identify a sequence of the peptide. 31. A method of sequencing peptides, each of which has a C-terminal amino acid (CTAA) and a N-terminal amino acid (NTAA), the method comprising: attaching peptide(s) to be sequenced to a solid substrate by their C-terminus, to form immobilized peptides; functionalizing the initial N-terminal amino acid of the immobilized peptides with a universal docking strand (DS) ssDNA oligo; contacting the DSs with an imaging strand (IS) oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule fluorescence lifetime (FLIM) measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; cleaving the initial N-terminal amino acid from the peptide to reveal a second N-terminal amino acid; and optionally, carrying out another cycle of analysis for the second N-terminal amino acid. 32. A method of sequencing peptides, each of which has a C-terminal amino acid (CTAA) and a N-terminal amino acid (NTAA), the method comprising: attaching peptide(s) to be sequenced to a solid substrate by their N-terminus, to form immobilized peptides; Filed: January 4, 2024 functionalizing the initial C-terminal amino acid of the immobilized peptides with a universal docking strand (DS) ssDNA oligo; contacting the DSs with an imaging strand (IS) oligo complementary to the DS oligo, which IS is conjugated to a first fluorophore; obtaining a single molecule fluorescence lifetime (FLIM) measurement for the first fluorophore for each peptide; optionally, repeating the single molecule FLIM measurement for one or more additional combinations of IS(s) and fluorophore(s) in the library; cleaving the initial C-terminal amino acid from the peptide to reveal a second C-terminal amino acid; and optionally, carrying out another cycle of analysis for the second C-terminal amino acid. 33. A method of sequencing a peptide, essentially as described herein. 34. The method of claim 33, wherein the method comprises detecting at least one spectral characteristic of a signal molecule, where the spectral characteristic is not fluorescence lifetime. 35. A kit for carrying out the method of any one of claims 1-34, comprising at least one pair of IS and DS. 36. The kit of claim 35, comprising at least two pairs of IS and DS, where the two pairs differ by the fluorophore contained in the IS or by sequence, or both.

Filed: January 4, 2024 37. A compound of Formula (II) or a salt, or solvate x is 0, 1 or 2; each R independently is selected from the group consisting of C₁-C₆ alkyl, -NO₂, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO₃, or any other common electron withdrawing groups; R¹ and R² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, -O-(C₁-C₆ alkyl), C₁-C₆ alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)- R, -O-C(=O)OR, -N-C(=S)NR, -N-(C=O)-OR, or any other common electron donating groups; y is 0, 1, 2, or 3; and each R³ independently is selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N- (C=O)-OR, or any other common electron donating groups. 38. The compound of claim 37 or a salt or solvate thereof, wherein x is 0. 39. The compound of claim 37 or a salt or solvate thereof, wherein y is 0. 40. The compound of claim 38 or a salt or solvate thereof, wherein y is 0. 41. The compound of any of claims 37-40, wherein: the C1-C6 alkyl of R¹ or R² is methyl, and the -O-(C1-C6 alkyl) of R¹ or R² is methoxy. Filed: January 4, 2024 42. The compound of claim 37, having the structure: ; wherein R₁ and R₂ are and OCH₃; with the proviso that R1 and R2 are the same; or a salt or solvate thereof. 43. The compound of claim 42, which is (4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. 44. A process for preparing a compound of Formula (I) Filed: January 4, 2024 or a salt or solvate thereof, comprising converting a compound of Formula (III) to a compound a Formula (II) or , and thereafter or solvate thereof to the compound of Formula (I) or a salt or solvate thereof, wherein: x is 0, 1 or 2; each R independently is selected from the group consisting of C1-C6 alkyl, -NO2, halogen, -C=OR, -C=SR, -C=ONR, -C=OOR, -SO3, or any other common electron withdrawing group; R¹ and R² are independently selected from the group consisting of hydrogen, C1-C6 alkyl, hydroxy, -O-(C1-C6 alkyl), halogen, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N- C(=S)NR, -N-(C=O)-OR, or any other common electron donating group; y is 0, 1, 2, or 3; and Filed: January 4, 2024 each R³ independently is selected from the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, halogens, -O-alkyl, -S-alkyl, -O-C(=O)R, -N-(C=O)-R, -O-C(=O)OR, -N-C(=S)NR, -N- (C=O)-OR, or any other common electron donating group. 45. The process of claim 44, wherein the compound of Formula (III) or the salt or solvate thereof is first converted to a compound of Formula (IV) or solvate thereof, followed by conversion of the compound of Formula (IV) or the salt or solvate thereof to the compound of Formula (II) or solvate thereof. 46. The process of claim 45, wherein the conversion of the compound of Formula (III) of the salt or solvate thereof to the compound of Formula (IV) or the salt or solvate thereof takes place by reacting carbon disulfide (CS₂) with the compound of Formula (III). 47. The process of claim 46, wherein the reaction takes place in the presence of a base. 48. The process of claim 47, wherein the base is a (C₁-C₆ alkyl)₃N. 49. The process of claim 48, wherein the (C₁-C₆ alkyl)₃N is triethylamine. 50. The process of claim 45, wherein the conversion of compound of Formula (IV) or the salt or solvate thereof to the compound of Formula (II) or the salt or solvate thereof takes place by Filed: January 4, 2024 reacting the compound of Formula (IV) or the salt or solvate thereof with di-tert-butyl carbonate (O-(C(=O)-OC(CH₃)₂)₂). 51. The process of claim 50, wherein the reaction takes place in the presence of one or more bases. 52. The process of claim 51, wherein the one or more bases comprise dimethyl aminopyridine (DMAP) and triethylamine. 53. The process of claim 45, wherein the compound has the structure: ; wherein R1 and R2 are and OCH3; with the proviso that R1 and R2 are the same; or a salt or solvate thereof. 54. The process of claim 53, wherein the compound is (4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)carbamothioic pivalic thioanhydride; or a salt or solvate thereof. 55. Use of the compound of any one of claims 37-54 in a peptide analysis as described herein.